Antibody and a method for producing an antibody that specifically binds to a full length amino acid sequence of human Delta-1

ABSTRACT

An antibody and a method for producing an antibody that specifically binds to a full length amino acid sequence of human Delta-1.

This application is a divisional of Ser. No. 09/995,593 filed Nov. 29,2001, now U.S. Pat. No. 7,141,379, which is a divisional of Ser. No.09/068,740 filed Jun. 18, 1998 now U.S. Pat. No. 6,337,387, which is theNational Stage of PCT/JP96/03356 filed Nov. 15, 1996, which claimspriority from Japanese Applications No. 7-299611 filed Nov. 17, 1995 andNo. 7-311811 filed Nov. 30, 1995.

BACKGROUND OF THE INVENTION

This invention relates to a novel bioactive substance which suppressesdifferentiation of undifferentiated cells.

DESCRIPTION OF THE RELATED ART

Human blood and lymph contain various types of cells and each cell playsimportant roles. For example, the erythrocyte carries oxygen; plateletshave hemostatic action; and lymphocytes prevent infection. These variouscells originate from hematopoietic stem cells in the bone marrow.Recently, it has been clarified that the hematopoietic stem cells aredifferentiated to various blood cells Osteoclasts and mast cells bystimulation of various cytokines in vivo and environmental factors. Inthe cytokines, there have been found, for example, erythropoietin (EPO)for differentiation to erythrocytes; granulocyte colony stimulatingfactor (G-CSF) for differentiation to leukocytes; and platelet growthfactor (mpl ligand) for differentiation to megakaryocytes which areplatelet producing cells, and the former two have already beenclinically applied.

The undifferentiated blood cells are generally classified into twogroups consisting of blood precursor cells which are destined todifferentiate to specific blood series and hematopoietic stem cellswhich have differentiation ability to all series and self-replicationactivity. The blood precursor cells can be identified by various colonyassays. However, identification method for the hematopoietic stem cellshave not been established. In these cells, stem cell factor (SCF),interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor(GM-CSF), interleukin-6 (IL-6), interleukin-1 (IL-1) granulocyte colonystimulating factor (G-CSF) and oncostatin M have been reported tostimulate cell differentiation and proliferation. Trials for expansionof hematopoietic stem cells in vitro have been examined in order toreplace bone marrow transplantation for applying hematopoietic stem celltransplantation therapy or gene therapy. However, when the hematopoieticstem cells are cultured in the presence of the above mentionedcytokines, multi-differentiation activities and self-replicationactivities, which are originally in the position of the hematopoieticstem cells, gradually disappeared and are changed to the blood cellprecursors which are only to differentiate to specific series after 5weeks of cultivation, and multi-differentiation activity which is one ofthe specific features of the hematopoietic stem cells, is lost (Wagneret al., Blood 86. 512–523, 1995).

For proliferation of the blood precursor cells, a single cytokine is notsufficient, but the synergistic action of several cytokines areimportant. Consequently, in order to proliferate the hematopoietic stemcells while maintaining specific features of the hematopoietic stemcells, it is necessary to add cytokines which suppress differentiationtogether with the cytokines which proliferate and differentiate theundifferentiated blood cells. In general, may cytokines which stimulateproliferation or differentiation of cells are known, but small numbersof cytokines which suppressed cell differentiation are known. Forexample, leukemia inhibitory factor (LIF) has an action of proliferationof mouse embryonic stem cells without differentiation, but it has noaction against the hematopoietic stem cells or blood precursor cells.Transforming growth factor (TGF-β) has suppressive action forproliferation against various cells, but no fixed actions against thehematopoietic stem cells or blood precursor cells.

Not only blood cells but also undifferentiated cells, especially stemcells, are thought to be involved in tissue regeneration. Theseregeneration of tissues and proliferation of undifferentiated cells ineach tissue can be applied in various ways by referring to the knownreference (Katsutoshi Yoshizato, Regeneration—a mechanism ofregeneration, 1996, Yodosha Publ. Co.).

Notch is a receptor type membrane protein which is involved inregulation of nerve cells differentiation found in Drosophia. Homologuesof the Notch are found in various animal kinds exceeding to theinvertebrate and vertebrate including nematoda (Lin-12). Xenopus laevis(Xotch), mouse (Motch) or human (TAN-2). Ligand of the Notch inDrosophila are known. These are Drosophila Delta (Delta) and DrosophilaSerrate (Serrate). Notch ligand homologues are found in various animalkinds as similar to the Notch of receptors (Artavanis-Tsakonas et al.,Science 268, 225–232, 1995).

Human Notch homologue, TAN-1 is found widely in the tissues in vivo(Ellisen et al., Cell 66. 649–661, 1991). Two Notch analogous moleculesother than TAN-1 are reported (Artavanis-Tsakonas et al., Science 268,225–232, 1995). Expression of TAN-1 was also observed in CD34 positivecells in blood cells by PCR (Polymerase Chain Reaction) (Milner et al.,Blood 83, 2057–2062, 1994). However, in relation to humans, gene cloningof human Delta and human Serrate, which are thought to be the Notchligand, have not been reported.

In Drosophila Notch, binding with the ligand was studied andinvestigated in detail, and it was found that the Notch can be bound tothe ligand with Ca** at the binding region, which is a repeated aminoacid sequence No. 11 and No. 12 in the amino acid sequence repeat ofEpidemal Growth Factor (EGF) like repeating (Fehon et al., Cell 61.523–534, 1990, Rebay et al., ibid. 67, 687–699, 1991 and Japan. PatentPCT Unexam. Publ. 7-503123). EGF-like repeated sequences are conservedin Notch homologues of the other species. Consequently, the samemechanism in binding with ligand is estimated. An amino acid sequencewhich is called DSL (Delta-Serrate-Lag-2) near the amino acid terminal,and EGF-like repeated sequence as in the receptor are conserved in theligand (Artavanis-Tsakonas et al.), Science 268, 225–232, 1995).

The sequence of DSL domain is not found except for the Notch ligandmolecules, and is specific to Notch ligand molecule. A common sequenceof DSL domain is shown in the sequence listing, SEQ ID NO: 1 in generalformula, and comparison with human Delta-1 and human Serrate-1 of thepresent invention and known Notch ligand molecules are shown in FIG. 1.

EGF-like sequence has been found in thrombomodulin (Jackman et al.,Proc. Natl. Acad. Sci. USA 83, 8834–8838, 1986), low density lipoprotein(LDL) receptor (Russell et al., Cell 37, 577–585, 1984), and bloodcoagulating factor (Furie et al., Cell 53, 505–518, 1988), and isthought to play important roles in extracellular coagulation andadhesion.

Recently, the vertebrate homologues of the cloned Drosophila Delta werefound in chicken (C-Delta-1) and Xenopus laevis (X-Delta-1), and it wasreported that X-Delta-1 had acted through Notch in the generation of theprotoneuron (Henrique et al., Nature 375, 787–790, 1995 and Chitnis etal., ibid. 375, 761–766, 1995). Vertebrate homologue of DrosophilaSerrate was found in rat as rat Jagged (Jagged) (Lindsell et al., Cell80, 909–917, 1995). According to the Lindsell et al., mRNA of the ratJagged is detected in the spinal cord of fetal rats. As a result ofcocultivation of a myoblast cell line is found. However, the rat Jaggedhas no action against the myoblast cell line without forced expressionof the rat Notch.

Considering the above reports, the Notch and ligand thereof may beinvolved in the differentiation regulation of the nerve cells, however,except for some myoblast cells, their actions against cells includingblood cells, especially primary cells, are unknown.

In the Notch ligand molecule, from the viewpoint of the prior studies onDrosophila and nematodae, the Notch ligand has specifically a structureof DSL domain which is not found other than in the Notch ligand.Consequently, the fact of having DSL domain means equivalent to ligandmolecule for the Notch receptor.

SUMMARY AND OBJECTS OF THE INVENTION

As mentioned above, concerning undifferentiated cells, proliferationthat maintains their specificies has not been achieved. Major reasonsare that factors suppressing differentiation of the undifferentiatedcells are not sufficiently known. An object of the present invention isto provide a compound originated from novel factors which can suppressdifferentiation of the undifferentiated cells.

We have set up a hypothesis that the Notch and its ligand have action ofdifferential regulation not only for neuroblasts and myoblasts but alsofor various undifferentiated cells, especially blood undifferentiatedcells. However, in case of clinical application in the humans, priorknown different species such as chicken or Xenopus laevos type notchligand have problems of species specificities and antigenicities.Consequently, to obtain previously unknown human Notch ligand isessentially required. We had an idea that a molecule having DSL domainand EGF-like domain which are common to Notch ligand molecules and aligand of the human Notch (TAN-1 etc.), which is a human Delta Homologue(hereinafter designated as human Delta) and human Serrate homologue(hereinafter designated as human Serrate), may be found. Also we have anidea that these findings may be a candidate for drugs useful fordifferential regulation of the undifferentiated cells, and we have triedto find out the same.

In order to find out human Notch ligands, we have analyzed amino acidsequences which are conserved in animals other than humans, and tried tofind out genes by PCR using mixed primers of the corresponding DNAsequence. As a result of extensive studies, we have succeeded inisolation of cDNAs coding amino acid sequences of two new molecules,novel human Delta-1 and novel human Serrate-1, and have prepared theexpression systems of protein having various forms using these cDNAs.Also we have established purification method of the proteins which werepurified and isolated.

Amino acid sequences of novel human Delta-1 are shown in the sequencelistings, SEQ ID NO: 2–4. DNA sequence coding these sequence is shown inthe sequence listing, SEQ ID NO: 8. Amino acid sequence of novel humanSerrate-1 is shown in the sequence listings, SEQ ID NO: 5–7. DNAsequence coding these sequence is shown in the sequence listing. SEQ IDNO: 10.

Physiological actions of the these prepared proteins were searched byusing nerve undifferentiated cells, preadipocytes, hepatocytes,myoblasts, skin undifferentiated cells, blood undifferentiated cells andimmuno undifferentiated cells. As a result, we have found that novelhuman Delta-1 and novel human Serrate-1 had an action ofdifferentiation-suppressive action to primary blood undifferentiatedcells, and had a physiological action to maintain undifferentiatedstate.

Such actions to the blood undifferentiated cells have never beenreported previously, and is a new discovery. No significant toxicactions were noted in the toxicity studies on mice, and usefulpharmaceutical effects were suggested. Consequently, the pharmaceuticalpreparations containing the molecule of the present invention, mediumcontaining the molecule of the present invention, and the device havingimmobilized thereon the molecule of the present invention are noveldrugs and medical materials which can maintain the bloodundifferentiated cells in the undifferentiated conditions. Antibodiesagainst human Delta-1 and human Serrate-1 are prepared by using antigensof the said human Delta-1 and human Serrate-1, and purification methodof the said antibodies are established. The present invention has beencompleted accordingly.

The present invention further related relates to a polypeptidecomprising amino acid sequence of SEQ ID NO: 1 of the sequence listingencoded in a gene of human origin, a polypeptide comprising at leastamino acid sequence of SEQ ID NO: 2 or NO: 5 of the sequence listing,the polypeptide comprising amino acid sequence of SEQ ID NO: 3 of thesequence listing, the polypeptide comprising amino acid sequence of SEQID NO: 4 of the sequence listing, the polypeptide comprising amino acidsequence of SEQ ID NO: 6 of the sequence listing, the polypeptidecomprising amino acid sequence of SEQ ID NO: 7 of the sequence listing,the polypeptide having differentiation suppressive action againstundifferentiated cells, the polypeptide in which undifferentiated cellsare undifferentiated cells other than those of the brain and nervoussystem or muscular system cells, and the polypeptide in whichundifferentiated cells are the undifferentiated blood cells. The presentinvention also relates to a pharmaceutical composition containing thepolypeptides, and the pharmaceutical composition in which use there isas a hematopoietic activator. The present invention further relates to acell culture medium containing the polypeptides, and the cell culturemedium in which the cell is the undifferentiated blood cell. The presentinvention still further relates to a DNA coding a polypeptide at leasthaving amino acid sequence of SEQ ID NO: 2 or NO: 5 of the sequencelisting, the DNA having DNA sequence 242–841 of SEQ ID NO: 8 or DNAsequence 502–1095 of SEQ ID NO: 10 of the sequence listing, the DNAcoding the polypeptide having amino acid sequence of SEQ ID NO: 3 of thesequence listing, the DNA coding the polypeptide having amino acidsequence of SEQ ID NO: 4 of the sequence listing, the DNA having DNAsequence 242–2347 of the SEQ ID NO: 8 of the sequence listing, the DNAcoding the polypeptide having amino acid sequence of SEQ ID NO: 6 of thesequence listing, the DNA having DNA sequence 502–3609 of SEQ ID NO: 10of the sequence listing, the DNA coding the polypeptide having aminoacid sequence of SEQ ID NO: 7 of the sequence listing, and the DNAhaving DNA sequence 502–4062 of SEQ ID NO: 10 of the sequence listing.The present invention still further relates to a recombinant DNA made byligating a DNA selected from the groups of DNA hereinabove and a vectorDNA which can express in the host cell, a cell transformed by therecombinant DNA, and a process for production of polypeptide byculturing cells and isolating the thus produced compound. The presentinvention still further relates to an antibody specifically recognizingthe polypeptide having the amino acid sequence of SEQ ID NO: 7 of thesequence listing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in details in the following:

Preparation of cDNA necessary for gene manipulation, expression analysisby Northern blotting, screening by hybridization, preparation ofrecombinant DNA, determination of DNA base sequence and preparation ofcDNA library, all of which are series of molecular biologicalexperiments, can be performed according to a description of theconventional textbook for the experiments. The above conventionaltextbook of the experiments is, for example, Maniatis et al. ed.Molecular Cloning, A laboratory manual, 1989, Eds., Sambrook, J.,Fritsch, E. F. and Maniatis, T., Cold Spring Harbor Laboratory Press.

A polypeptide of the present invention has at least one of thepolypeptides in the sequence listing SEQ ID NO: 1–7. A mutant and allelewhich naturally occur in the nature are included in the polypeptide ofthe present invention unless the polypeptides of the sequence listing,SEQ ID NO: 1–7 lose their properties. Modification and substitution ofamino acids are described in details in the patent application by thename of Benntt et al. (National Unexam. Publ. WO96/2645) and can beprepared according to the description thereof.

A DNA sequence coding polypeptides of the sequence listing, SEQ ID NO:2–4 is shown in the sequence listing, SEQ ID NO: 8, and a DNA sequencecoding polypeptides of the sequence listing, SEQ ID NO: 5–7 is show inthe sequence listing, SEQ ID NO: 10, together with their amino acidsequences. In these DNA sequences, even if amino acid level mutation isnot generated, naturally isolated chromosomal DNA or cDNA thereof mayhave a possibility to mutate in the DNA base sequence as a result ofdegeneracy of genetic code without changing amino acid sequence coded bythe DNA. A 5′-untranslated region and 3′-untranslated region are notinvolved in amino acid sequence determination of the polypeptide, so DNAsequences of these regions are easily mutated. The base sequenceobtained by these degeneracies of genetic codes is included in the DNAof the present invention.

Undifferentiated cells in the present invention are defined as cellswhich can grow by specific stimulation, and cells which can bedifferentiated to the cells having specific functions as a result of thespecific stimulations. These include undifferentiated cells of the skintissues, undifferentiated cells of the brain and nervous systems,undifferentiated cells of the muscular systems and undifferentiatedcells of the blood cells. These cells include the cells ofself-replication activity which are called stem cells, and the cellshaving an ability to generate the cells of these lines. Thedifferentiation-suppressive action means suppressive action forautonomous or heteronomous differentiation of the undifferentiatedcells, and is an action for maintaining undifferentiated condition. Thebrain and nervous undifferentiated cells can be defined as cells havingability to differentiate to the cells of the brain or nerve havingspecific functions by specific stimulation. The undifferentiated cellsof the muscular systems can be defined as cells having ability todifferentiate to the muscular cells having specific functions byspecific stimulation. The blood undifferentiated cells in the presentinvention can be defined as cell groups consisting of the bloodprecursor cells which are differentiated to the specific blood seriesidentified by blood colony assay, and hematopoietic stem cells havingdifferentiation to every series and self-replication activities.

In the sequence listing, amino acid sequence in SEQ ID NO: 1 showsgeneral formula of common amino acid sequence of DSL domain which is acommon domain structure of the Notch ligand molecules, and at least thisdomain structure corresponds to the sequence listing, AMINO ACIDS158–200 of the human Delta-1, or the sequence listing AMINO ACIDS156–198 of the human Serrate-1.

The amino acid sequence in the sequence listing, SEQ ID NO: 2 is asequence of the active center of the present invention of human Delta-1minus the signal peptide, i.e. amino acid sequence from the aminoterminal to DSL domain, and corresponds to an amino acid No. 1 to 200 inSEQ ID NO: 4 of the mature full length amino acid sequence of humanDelta-1 of the present invention. The amino acid sequence in SEQ ID NO:3 is amino acid sequence of extracellular domain of the presentinvention of human Delta-1 minus the signal peptide, and corresponds toan amino acid No. 1 to 520 in SEQ ID NO: 4 of the mature full lengthamino acid sequence of human Delta-1 of the present invention. The aminoacid sequence of SEQ ID NO: 4 is the mature full length amino acidsequence of the human Delta-1 of the present invention.

The amino acid sequence in the sequence listing, SEQ ID NO: 5 is asequence of the active center of the present invention of humanSerrate-1 minus the signal peptide, i.e. amino acid sequence from theamino terminal to DSL domain, and corresponds to an amino acid No. 1 to198 in SEQ ID NO: 7 of the mature full length amino acid sequence ofhuman Serrate-1 of the present invention. The amino acid sequence in SEQID NO: 6 is amino acid sequence of extracellular domain of the presentinvention of human Serrate-1 minus the signal peptide, and correspondsto an amino acid No. 1 to 1036 in SEQ ID NO: 7 of the mature full lengthamino acid sequence of human Serrate-1 of the present invention. Theamino acid sequence of SEQ ID NO: 7 is the mature full length amino acidsequence of the human Serrate-1 of the present invention.

The sequence of SEQ ID NO: 8 is the total amino acid sequence of humanDelta-1 of the present invention and cDNA coding the same, and thesequence of SEQ ID NO: 10 is total amino acid sequence of humanSerrate-1 of the present invention and cDNA coding the same.

The left and right ends of the amino acid sequences in the sequencelisting indicate amino terminal (hereinafter designated as N-terminal)and carboxyl terminal (hereinafter designated as C-terminal),respectively, and the left and right ends of the nucleotide sequencesare 5′-terminal and 3′-terminal, respectively.

Cloning of human Notch ligand gene can be performed by the followingmethod. During the evolution of the organisms, a part of amino acidssequences of the human Notch ligand is conserved. DNA sequencecorresponding to the conserved amino acid sequence is designed, and isused as a primer of RT-PCR (Reverse Transcription Polymerase Chainreaction), then a PCR template of the human origin is amplified by PCRreaction, thereby fragments of human Notch ligand can be obtainable.Furthermore, RT-PCR primer is prepared by applying the known DNAsequence information of the Notch ligand homologue of the organismsother than humans, and the known gene fragments can be possibly obtainedfrom PCR template of the said organisms.

In order to perform PCR for obtaining fragments of human Notch ligand,PCR for DSL sequence is considered, but a large number of combinationsof DNA sequence corresponding to amino acid sequence conserved in thisregion can be expected, and a design for PCR is difficult. As a result,PCR of the EGF-like sequence has to be selected. As explainedhereinbefore, since EFG-like sequence is conserved in a large number ofmolecules, to obtain the fragments and identification are extremelydifficult.

We have designed and prepared about 50 PCR primer sets, for example, theprimer set of the sequence shown in Example 1, PCR was performed withthese primer sets by using PCR template of cDNA prepared from poly A+RNA of various tissues of human origin, and more than 10 PCR productsfrom each tissue were subcloned, as well as performing sequencing formore than 500 types. A clone having a desired sequence could beidentified. Namely, the obtained PCR product is cloned in the cloningvector, transforming the host cells by using recombinant plasmid whichcontains the PCR product, culturing the host cells containing therecombinant plasmid on a large scale, purifying and isolating therecombinant plasmid, checking the DNA sequence of PCR product which isinserted into the cloning vector, and trying to obtain the gene fragmentwhich may have a sequence of human Delta-1 by comparing with thesequence of the known Delta of other species. We have succeeded to findout the gene fragment which contains a part of cDNA of human Delta-1,the same sequence of DNA sequence from 1012 to 1375 described in thesequence listing, SEQ ID NO: 8.

We have also designed and prepared about 50 PCR primer sets, forexample, the primer set of the sequence shown in Example 3, and PCR wasperformed with these primer sets by using PCR template of cDNA preparedfrom poly A+ RNA of various tissues of human origin, and more than 10PCR products from each tissue were subcloned, as well as performingsequencing for more that 500 types. A clone having a desired sequencecould be identified. Namely, the obtained PCR product is cloned in thecloning vector, transforming the host cells by using recombinant plasmidwhich contains the PCR product, culturing the host cells containing therecombinant plasmid on a large scale, purifying and isolating therecombinant plasmic, checking the DNA sequence of PCR product which isinserted into the cloning vector, and trying to obtain the gene fragmentwhich may have a sequence of human Serrate-1 by comparing with thesequence of the known Serrate of other species. We have succeeded tofind out the gene fragment which contains a part of cDNA of humanSerrate-1, the same sequence of DNA sequence from 1272 to 1737 describedin the sequence listing, SEQ ID NO: 10.

A full length of the objective gene can be obtained form the humangenomic gene library or cDNA library by using the thus obtained humanDelta-1 fragment or human Serrate-1 gene fragment. The full lengthcloning can be made by isotope labeling and non-isotope labeling withthe partial cloning gene, and screening the library by hybridization orother method. Isotope labeling can be performed by, for example,terminal labeling by using [³²P] γ-ATP and T4 polynucleotide kinase, orother labeling methods such as nick translation or primer extensionmethod can be applied. In another method, human originated cDNA libraryis ligated into the expression vector, expressing by COS-7 or othercells, and screening the objective gene by expression cloning to isolatecDNA of the ligand. In the expression cloning, a cell sorterfractionation method which is applied with binding with polypeptidecontaining amino acid sequence of prior known 4 Notches such as TAN-1,and a detection method by film emulsion using radioisotope can bementioned. In this specification, methods for obtaining genes of humanDelta-1 and human Serrate-1 are explained, and in addition to thatobtaining the Notch ligand homologue gene of the other organism isimportant for analysis of ligand action. This may be made by the sametreatment. The obtained gene is subjected to DNA sequence determinationand amino acid sequence can be estimated.

As shown in Example 2, gene fragments containing human Delta-1 PCRproduct are labeled with radioisotope to prepare hybridization probescreening is performed using cDNA of human placenta origin as thescreening library. DNA sequences of the thus obtained clones aredetermined, and the clone is obtained containing DNA nucleotide sequenceshown in the sequence listing. SEQ ID NO: 8, and shown to have the aminoacid sequence coded in the sequence listing. SEQ ID NO: 4. We havesucceeded in cloning cDNA coding fill length of amino acids sequence ofhuman Delta-1.

These sequences were compared with the data base (Genbank release 89,June, 1995), and found that these were novel sequences. The said aminoacid sequence was analyzed in hydrophilic part and hydrophobic partaccording to a method by Kyte-Doolittle (J. Mol. Biol. 157: 105, 1982).A result indicated that human Delta-1 of the present invention isexpressed on cells as a cellular membrane protein having a transmembranedomain.

As shown in Example 4, gene fragments containing human Serrate-1 PCRproduct are labeled with radioisotope to prepare hybridization probe,screening is performed using cDNA of human placenta origin as thescreening library, DNA sequences of the thus obtained clones aredetermined and the clone is obtained containing DNA nucleotide sequenceshown in the sequence listing, SEQ ID NO: 10, and shown to have theamino acid sequence coded in the sequence listing, SEQ ID NO: 7. In thisscreening, an intracellular part of gene sequence coding a full lengthof amino acids sequence, namely a peripheral part of termination codoncannot be cloned. Consequently, as shown in Example 4, gene cloning isperformed by RACE method (rapid amplification of cDNA ends, Frohman etal., Proc. Nati. Acad. Sci. U.S.A. 85, 8998–9002, 1988) and finallysucceeded in cloning of cDNA coding full length of amino acid sequenceof human Serrate-1.

These sequences were compared with the data base (Genbank release 89,June, 1995, and found that these were novel sequences. The said aminoacid sequence was analyzed in hydrophilic part and hydrophobic partaccording to a method by Kyte-Doolittle (J. Mol. Biol. 157: 105, 1982).A result indicated that human Serrate-1 of the present invention isexpressed on cells as a cellular membrane protein having a transmembranedomain.

Examples of plasmids integrated with cDNA are, for example, E. colioriginated pBR322, pUC18, pUC19, pUC118 and pUC119 (Takara Shuzo Co.,Japan), but other plasmids can be used if they can replicate andproliferate in the host cells. Examples of phage vectors integrated withcDNA are, for example, λgt10 and λgt11, but other vectors can be used ifthey can grow in the host cells. The thus obtained plasmids aretransducted into suitable host cells such as genus Escherichia and genusBacillus using calcium chloride method. Examples of the above genusEscherichia are Eseherichia coli K12HB101, MC1061, LE392 and HM109.Example of the above genus Bacillus is Bacillus subtilis MI114. Phagevector can be introduced into the proliferated E. coli by the in vitropackaging method (Enquist and Sternberg, Meth. Enzymol., 68, 281-,1979).

According to the analysis of amino acid sequence of the human Delta-1,amino acid sequence of a precursor of human Delta-1 consists of 723amino acids residue shown in the sequence listing, SEQ ID NO: 8, and thesignal peptide domain is estimated to correspond to an amino acidsequence of 21 amino acids residue from No. −21 methionine to No. −1serine of the sequence listing; extracellular domain: 520 amino acidsresidue from No. 521 proline to No. 552 leucine; and intracellulardomain: 150 amino acids region from No. 553 glutamine to No. 702 valine.These domains are estimated domain construction from amino acidsequences, and actual presence form may differ from the above structure,and constitutional amino acids of each domain hereinabove defined mayhave possibility to change 5 to 10 amino acids sequence.

According to a comparison in amino acid sequence of human Delta-1 andDelta homologue of the other organisms, the homologies with DrosophilaDelta, chick Delta and Xenopus laevis are 47.6%, 83.3% and 76.2%,respectively. The human Delta-1 of the present invention is differentfrom these Deltas and is novel substance which is clarified at first bythe present inventors. Search from all of organisms in the above database indicated that polypeptides having the identical sequence of thehuman Delta-1 could not be found.

The homologues of Notch ligand have evolutinally conserved commonsequence, i.e. repeated DSL sequence and EGF-like sequence: As a resultof comparison with amino acid sequence of human Delta-1, these conservedsequence is estimated. Namely, DSL sequence corresponds to 43 aminoacids residue from No. 158 cysteine to No. 200 cysteine of the aminoacid sequence in the sequence listing, SEQ ID NO: 4. EGF-like sequenceexists with 8 repeats wherein, in the amino acid sequence in thesequence listing SEQ ID NO: 4, the first EGF-like sequence from No. 205cysteine to No. 233 cysteine; the second EGF-like sequence from No. 236cysteine to No. 264 cysteine; the third EGF-like sequence from No. 271cysteine to No. 304 cysteine; the fourth EGF-like sequence from No. 311cysteine to No. 342 cysteine; the fifth EGF-like sequence from No. 349cysteine to No. 381 cysteine; the sixth EGF-like sequence from No. 388cysteine to No. 419 cysteine; the seventh EGF-like sequence from No. 426cysteine to No. 457 cysteine; and the eighth EGF-like sequence from No.464 cysteine to No. 495 cysteine.

A part of sugar chain attached is estimated from amino acid sequence ofthe human Delta-1 may be No. 456 asparagine residue in the sequencelisting, SEQ ID NO: 4 as a possible binding site of N-glycoside bondingfor N-acetyl-D-glucosamine. O-glycoside bond of N-acetyl-D-galactosamineis estimated to be a serine or threonine residue rich part. Proteinbound with sugar chain is generally thought to be stable in vivo and tohave strong physiological activity. Consequently, in the amino acidsequence of polypeptide having sequence of the sequence listing, SEQ IDNO: 2, 3 or 4, polypeptides having N-glucoside or O-glucoside bond withsugar chain of N-acetyl-D-glucosamine or N-acetyl-D-galactosamine isincluded in the present invention.

According to the analysis of amino acid sequence of the human Serrate-1,amino acid sequence of a precursor of human Serrate-1 consists of 1218amino acids residue shown in the sequence listing, SEQ ID NO: 10, andthe signal peptide domain is estimated to correspond 31 amino acidsresidue in the amino acid sequence from No. −31 methionine to No. −1alanine of the sequence listing; extracellular domain: 10366 amino acidsresidue from No. 1 serine to No. 1036 asparagine; transmembrane domain:26 amino acids residue from No. 1037 phenylalanine to No. 1062 leucine;and intracellular domain: 106 amino acids domain from No. 1063 arginineto No. 1187 valine. These domains are estimated domain construction fromamino acid sequences, and actual presence form may differ from the abovestructure, and constitutional amino acids of each domain hereinabovedefined may have possibility to change 5 to 10 amino acids sequence.

According to a comparison in amino acid sequence of human serrate-1 andSerrate homologue of the other organisms, the homologies with DrosophilaSerrate, and rat Jagged are 32.1% and 95.3%, respectively. The humanSerrate-1 of the present invention is different from these Serrates andis novel substance which is clarified at first by the present inventors.Search from all of organisms in the above data base indicated thatpolypeptides having the identical sequence of the human Serrate-1 couldnot find out.

The homologues of Notch ligand have evolutionally conserved commonsequence, i.e. repeated DSL sequence and EGF-like sequence. As a resultof comparison with amino acid sequence of human Serrate-1 and otherNotch ligand homologues, these conserved sequence is estimated. Namely,DSL sequence corresponds to 43 amino acids residue from No. 156 cysteineto No. 198 cysteine of the amino acid sequence in the sequence listing,SEQ ID NO: 7. EGF-like sequence exists with 16 repeats wherein, in theamino acid sequence in the sequence listing, SEQ ID NO: 7, the firstEGF-like sequence from No. 205 cysteine to No. 231 cysteine; the secondEGF-like sequence from No. 234 cysteine to No. 262 cysteine; the thirdEGF-like sequence from No. 269 cysteine to No. 302 cysteine; the fourthEGF-like sequence from No. 309 cysteine to No. 340 cysteine; the fifthEGF-like sequence from No. 356 cysteine to No. 378 cysteine; the sixthEGF-like sequence from No. 423 cysteine to No. 453 cysteine; the eighthEGF-like sequence from No. 462 cysteine to No. 453 cysteine; the ninethEGF-like sequence from No. 498 cysteine to No. 529 cysteine; the 10^(th)EGF-like sequence from No. 536 cysteine to No. 595 cysteine; the 11thEGF-like sequence from No. 602 cysteine to No. 633 cysteine; the 12^(th)EGF-like sequence from No. 640 cysteine to No. 671 cysteine; the 13^(th)EGF-like sequence from No. 678 cysteine to No. 709 cysteine; the 14^(th)EGF-like sequence from No. 717 cysteine to No. 748 cysteine; and the16^(th) EGF-like sequence from No. 793 cysteine to No. 824 cysteine.However, the 10^(th) EGF-like sequence has irregular sequence containing10 residues of cysteine.

A part of sugar chain attached is estimated from amino acid Sequence ofthe human Serrate-1 may be No. 112, 131, 186, 351, 528, 554, 714, 1014and 1033 asparagine residue in the sequence listing. SEQ ID NO: 7 as apossible binding site of N-glycoside bonding for N-acetyl-D-glycosamine.O-glycoside bond of N-acetyl-D-galactosamine is estimated to be a serineor threonine residue rich part, Protein bound with sugar chain isgenerally thought to be stable in vivo and to have strong physiologicalactivity. Consequently, in the amino acid sequence of polypeptide havingsequence of the sequence listing, SEQ ID NO: 5, 6 or 7, polypeptideshaving N-glucoside or O-glucoside bond with sugar chain ofN-acetyl-D-glucosamine or N-acetyJ-D-galactosamine is included in thepresent invention.

As a result of studies on binding of Drosophila Notch and its ligand,amino acid region necessary for binding with ligand of Drosophila Notchwith the Notch is from N-terminal to DSL sequence of the maturedprotein, in which signal peptide is removed (Japan. Pat. PCT Unexam.Publ. No. 7-503121). This fact indicates that a domain necessary forexpression of ligand action of human Notch ligand molecule is at leastthe DSL domain. i.e. a domain containing amino acid sequence of thesequence listing, SEQ ID NO: 1, and a domain at least necessary forexpression of ligand action of human Delta-1 is novel amino acidsequence shown in the sequence listing, SEQ ID NO: 2, and further adomain at least necessary for expression of ligand action of humanSerrate-1 is novel amino acid sequence shown in the sequence listing,SEQ ID NO: 5.

An mRNA of human Delta-1 can be detected by using DNA coding a part orall of gene sequence in the sequence listing, SEQ ID NO: 8, and an mRNAof human Serrate-1 can be detected by using DNA coding a part or all ofgene sequence in the sequence listing, SEQ ID NO: 10. For example, amethod for detection of expression of these genes can be achieved byapplying with hybridization or PCR by using complementary nucleic acidsof above 12 mer or above 16 mer, preferably above 18 mer having nucleicacid sequence of a part of sequence in the sequence listing, SEQ ID NO:8 or 10, i.e. antisense DNA or antisense RNA, its methylated,methylphosphated, deaminated, or thiophosphated derivatives. By the samemethod, detection of homologues of the gene of other organisms such asmice or gene cloning can be achieved. Further cloning of genes in thegenome including humans can be made. Using these genes cloned by suchlike methods, further detailed functions of the human Delta-1 or humanSerrate-1 of the present invention can be clarified. For example, usingthe modern gene manipuration techniques, every methods includingtransgenic mouse, gene targeting mouse or double knockout mouse in whichgenes relating to the gene of the present invention are inactivated, canbe applied. If abnomalities in the genome of the present gene is found,application to gene diagnosis and gene therapy can be made.

A transformant in which vector pUCDL-IF, which contains cDNA codingtotal animo acid sequence of human Delta-1 of the present invention, istransformed into E.coli JM109, has been deposited in the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, MITI, of 1-1-3, Higasi, Tsukuba-shi,Ibaragi-ken, Japan, as E. coli: JM109-pUCDL-1F. Date of deposit was Oct.28, 1996, and deposition No. is FBRM BP-5728. A transformant in whichvector pUCSR-1, which contains cDNA coding total animo acid sequence ofhuman Serrate-1 of the present invention, is transformed into E.coliJM109, has been deposited in the National Institute of Bioscience andHuman-Technology, Agency of industrial Science and Technology, MITI, of1-1-3, Higasi, Tsukuba-shi, Ibaragi-ken, Japan, as E. coli:JM109-pUCSR-1. Date of deposit was Oct. 28, 1996, and deposition No. isFBRM BP-5726.

Exprssion and purification of various forms of human Delta-1 and humanSerrate-1 using cDNA coding amino acid sequence of human Delta-1 andhuman Serrate-1 isolated by the above methods are known in thereferences (Kriegler, Gene Transfer and Expression—A Laboratory ManualStockton Press, 1990 and Yokota et al. Biomanual Series 4, Gene transferand expression and analysis, Yodosha Co., 1994). A cDNA coding the aminoacid sequence of the isolated said human Delta-1 and human Serrate-1 isligated to preferable expression vector and is produced in the hostcells of eukaryotic cells such as animal cells and insect cells orprokaryotic cells such as bacteria.

In the expression of human Delta-1 and human Serrate-1 of the presentinvention. DNA coding polypeptide of the present invention may have thetranslation initiation codon in 5′-terminal and translation terminationcodon in 3′-terminal. These translation initiation codon and translationtermination codon can be added by using preferable synthetic DNAadapter. Further for expression of the said DNA, promoter is linkaged inthe upstream of the DNA sequence. Examples of vector are plasmidoriginated from Bacullus, plasmid originated from yeast or bacteriophagesuch as λ-phage and animal virus such as retrovirus and vaccinia virus.

Examples of promoters used in the present invention are any promoterspreferable for corresponding to the host cells used in gene expression.

In case that the host cell in the transformation is genus Eseherichia,tac-promoter, trp-promoter and lac-promoter are preferable, and in caseof host of genus Bacullus, SP01 promoter and SP02 promoter arepreferable, and in case of host of yeast, PGK promoter, GAP promoter andADH promoter are preferable.

In case that the host cell is animal cells, a promoter originated fromSV40 such as SRα promoter as described in Example 51 promoter ofretrovirus, metallothionein promoter and heatshock promoter can beapplied.

Polypeptide of the present invention can be expressed by using theexpression vector having ability to be used by any person skilled in thearts.

Expression of the polypeptide of the present invention can be made byusing only DNA coding the amino acid sequence of the sequence listing,SEQ ID NO: 2, 3, 4, 5, 6 or 7. However, the protein added with specificfunction can be produced by using DNA, to which added cDNA coding theknown antigen epitope for easier detection of the produced polypeptideor added cDNA coding the immunoglobulin Fc for forming multimer.

As shown in Example 5, we have prepared expression vectors, whichexpress extracellular proteins of human Delta-1, as follows:

-   -   1) DNA coding the amino acids from No. 1 to 520 in amino acid        sequence in the sequence listing, SEQ ID NO: 3,    -   2) DNA coding chimera protein to which added polypeptide having        8 amino acid, i.e. an amino acid sequence consisting of Asp Tyr        Lys Asp Asp Asp Asp Lys (hereinafter, designates FLAG sequence,        the sequence listing, SEQ ID NO: 12), in the C-terminal of the        amino acids from No. 1 to 520 in amino acid sequence in the        sequence listing, SEQ ID NO: 3, and    -   3) DNA coding chimera protein to which added Fc sequence below        the hinge region of human IgG1 (refer to International Patent        Publ. WO96/11221 in the C-terminal of the amino acids from No. 1        to 520 in amino acid sequence in the sequence listing, SEQ ID        NO:3, and to have dimer structure by disulfide bond in the hinge        region,        are ligated individually with the expression vector pMKITNeo        (Maruyama et al. Japan Molecular Biology Soc. Meeting        Preliminary lecture record, obtainable from Dr. Maruyama in        Tokyo Medical and Dental College, containing promoter SRα) to        prepare extracellular expression vectors of human Delta-1.

The full-length expression vectors of the human Delta-1 as theexpression vectors, which express full-length proteins of the humanDelta-1, can be prepared as follows.

-   -   4) DNA coding amino acids from No. 1 to 702 in the sequence        listing, SEQ ID NO: 4 and    -   5) DNA coding chimera protein to which added polypeptide having        FLAG sequence in the C-terminal of amino acids from No. 1 to 702        in the sequence listing, SEQ ID NO: 4        are ligated individually with the expression vector pMKITNeo to        prepare the full-length expression vectors of human Delta-1. The        transformant is prepared by using expression plasmid containing        DNA coding the thus constructed said human Delta-1.

As shown in Example 6, we have prepared expression vectors, whichexpress extracellular proteins of human Serrate-1, as follows.

-   -   6) DNA coding the amino acids from No. 1 to 1036 in amino acid        sequence in the sequence listing, SEQ ID NO: 6,    -   7) DNA coding chimera protein to which added polypeptide having        FLAG sequence in the C-terminal of the amino acids from No. 1 to        1036 in amino acid sequence in the sequence listing, SEQ ID NO:        6, and    -   8) DNA coding chimera protein to which added said Fc sequence in        the C-terminal of the amino acids from No. 1 to 1036 in amino        acid sequence in the sequence listing, SEQ ID NO: 6, and to have        dimer structure by disulfide bond in the hinge region,        are ligated individually with the expression vector pMKITNeo to        prepare extracellular expression vectors of human Serrate-1.

The full-length expression vectors of the human Serrate-1 as theexpression vectors, which express full-length proteins of the humanSerrate-1, can be prepared as follows.

-   -   9) DNA coding amino acids from No. 1 to 1187 in the sequence        listing, SEQ ID NO: 7 and    -   10) DNA coding chimera protein to which added polypeptide having        FLAG sequence in the C-terminal of amino acids from No. 1 to        1187 in the sequence listing, SEQ ID NO: 7        are ligated individually with the expression vector pMKITNeo to        prepare the full-length expression vectors of human Serrate-1.        The transformant is prepared by using expression plasmid        containing DNA coding the thus constructed said human Serrate-1.

Examples of the host are genus Escherichia, genus Bacullus, yeast andanimal cells. Examples of animal cells are simian cell COS-7 and Vero,Chinese hamster cell CHO and silk worm cell SF9.

As shown in Example 7, the above expression vectors 1)–10) aretransduced individually: the human Delta-1 or human Serrate-1 areexpressed in COS-7 cell (obtainable from the Institute of Physical andChemical Research, Cell Development Bank, RCB0539), and thetransformants which were transformed by these expression plasmids, canbe obtained. Further, human Delta-1 polypeptide and human Serrate-1polypeptide can be produced by culturing the transformants underpreferable culture condition in medium by known culture method.

As shown in Example 8, human Delta-1 polypeptide and human Serrate-1polypeptide can be isolated and purified from the above cultured mass,in general, by the following methods.

For extraction of the substance from cultured microbial cells or cells,microbial cells or cells are collected by known method such ascentrifugation after the cultivation, suspended in preferable buffersolution, disrupted the microbial cells or cells by means ofultrasonication, lysozyme and/or freeze-thawing and collected crudeextract by centrifugation or filtration. The buffer solution may containprotein-denaturing agents such as urea and guanidine hydrochloride orsurface active agents such as Triton-X. In case of secretion in thecultured solution, the cultured mass is separated by the known methodsuch as centrifugation to separate from microbial cells or cells and thesupernatant solution is collected.

The thus obtained human Delta-1 or human Serrate-1, which are containedin the cell extracts or cell supernatants, can be purified by knownprotein purification methods. During the purification process, forconfirmation of existence of the protein, in case of the fused proteinsof the above FLAG and human IgGFc, they can be detected by immunoassayusing antibody against known antigen epitope and can be purified. Incase of not to express as such the fused protein, the antibody inExample 9 can be used for detection.

Antibodies, which specifically recognize human Delta-1 and humanSerrate-1, can be prepared as shown in Example 9. Antibodies can beprepared by the methods described in the reference (Antibodies alaboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory) orrecombinant antibodies expressed in cells by using immunoglobulin genesisolated by gene cloning method. The thus prepared antibodies can beused for purification of human Delta-1 and human Serrate-1. The humanDelta-1 or human Serrate-1 can be detected and assayed by usingantibodies which recognize specifically human Delta-1 or human Serrate-1as shown in Example 9, and can be used for diagnostic agents fordiseases accompanied with abnormal differentiation of cells such asmalignant tumors.

More useful purification method is the affinity chromatography usingantibody. Antibodies used in this case are antibodies described inExample 9. For fused protein, antibodies against FLAG in the case ofFLAG, and protein G or protein A in the case of human IgGFc as shown inExample 8.

Any fused protein other than the protein as shown hereinabove can beused. For example, histidine Tag and myc-tag can be mentioned. Any fusedproteins can be prepared by using methods of present day geneticengineering techniques other than the known methods, and peptides of thepresent invention derived from those fused proteins are in the scope ofthe present invention.

Physiological functions of the thus purified human Delta-1 and humanSerrate-1 proteins can be identified by various assay methods, forexample, physiological activity assaying methods using cell lines andanimals such as mice and rats, assay methods of intracellular signaltransduction based on molecular biological means, binding with Notchreceptor etc.

We have observed actions for blood undifferentiated cells by using IgG1chimera proteins of human Delta-1 and human Serrate-1.

As a result, we have found that, as shown in Example 10, in theumbilical cord blood derived blood undifferentiated cells, in which CD34positive cell fraction is concentrated, polypeptides of the presentinvention have suppressive action of colony forming action against bloodundifferentiated cells, which shows colony formation in the presence ofcytokines. The suppressive action is only observed in the presence ofSCF. This kind of effect has never been known.

As shown in Example 11, we have found that a maintenance of colonyforming cells is significantly extended by addition of IgG1 chimeraprotein of human Delta-1 or human Serrate-1 in the long term (8 weeks)liquid culture in the presence of cytokines such as SCF, IL-3, IL-6,GM-CSF and Epo. Further we have found that the polypeptides of thepresent invention had an action not to suppress growth of the colonyforming cells. A cytokine, MIP-1α having migration and differentiationsuppressive action of blood cells (Verfaillie et al., J. Exp. Med. 179,643–649. 1994), has no action for maintaining undifferentiation forblood undifferentiated cells.

Further as shown in Example 12, we have found that as a result of addingIgG1 chimera protein of human Delta-1 or human Serrate-1 to the liquidculture in the presence of cytokines, the human Delta-1 and humanSerrate-1 had activities for significantly maintaining LTC-IC (Long-TermCulture-Initiating Cells) number, which is positioned mostundifferentiated blood stem cells in the human blood undifferentiatedcells.

These results indicate that the human Delta-1 and human Serrate-1suppress differentiation of blood undifferentiated cells, and theseactions spread from blood stem cells to colony forming cells. Thesephysiological actions are essential for in vitro expansion of bloodundifferentiated cells. Cells cultured in the medium containing humanDelta-1 or human Serrate-1 are efficient in recovery of suppresion ofbone marrow after administration of antitumor agents, accordingly invitro growth of hemopoietic stem cells may be possible if otherconditions would be completed. Further pharmaceuticals containing thepolypeptide of the present invention have action protection and releaseof the bone marrow suppressive action, which is observed in adverseeffects of antitumor agents.

Suppressive action for differentiation of cells in the undifferentiatedcells other than blood cells is expected and stimulating action fortissue regeneration can be expected.

In the pharmaceutical use, polypeptides of the present invention arelyophilized with adding preferable stabilizing agents such as humanserum albumin, and is used in dissolved or suspended condition withdistilled water for injection when it is in use. For example,preparation for injection or infusion at the concentration of 0.1–1000μg/ml may be provided. A mixture of the compound of the presentinvention 1 mg/ml and human serum albumin 1 mg/ml divided in a vialcould maintain activity of the said compound for long term. Forculturing and activating cells in vitro, lyophilized preparation orliquid preparation of the polypeptide of the present invention areprepared and are added to the medium or immobilized in the vessel forculture. Toxicity of the polypeptide of the present invention wastested. Any polypeptide, 10 mg/kg was administered intraperitoneally inmice, but no death of mice was observed.

In vitro physiological activity of the polypeptide of the presentinvention can be evaluated by administering to disease model mice or itsresembled disease rats or monkeys, and examining recovery of physicaland physiological functions and abnormal findings. For example, in caseof searching abnormality in relation to hemopoietic cells, bone marrowsuppressive model mice are prepared by administering 5-FU series ofantitumor agents, and bone marrow cell counts, peripheral blood cellcounts and physiological functions are examined in the administeredgroup or the non administered group of mice. Further, in case ofsearching in vitro cultivation and growth of hemopoieticundifferentiated cells including hemopoietic stem cells, the bone marrowcells of mice are cultured in the groups with or without addition of thecompound of the present invention, and the cultured cells aretransferred into the lethal dose irradiated mice. Result of recovery isobserved with the indications of survival rate and variation of bloodcounts. These results can be extrapolated to the humans, and accordinglyuseful effective data for evaluation of the pharmacological activitiesof the compound of the present invention can be obtained.

Applications of the compound of the present invention forpharmaceuticals include diseases with abnormal differentiation of cells,for example leukemia and malignant tumors. These are cell therapy, whichis performed by culturing human derived cells in vitro while maintainingtheir original functions or adding new functions, and a therapy, whichis performed by regenerating without damaging the functions originallyexisting in the tissues by administering the compound of the presentinvention under the regeneration after tissue injury. Amount ofadministration may differ in the type of preparation and ranges from 10μg/kg to 10 mg/kg.

Further strong physiological activity can be achieved by expression offorming multimer of the polypeptide of the present invention.

As shown in Example 10, since the suppressive action of human Delta-1and human Serrate-1 is stronger in the IgG chimera protein having dimerstructure, a form of stronger physiological activity is preferablyexpressed in the form of multimer formation.

Human Delta-1 and human Serrate-1 having multimer structure can beproduced by a method of expressing chimera protein with human IgG Fcregion as described in the example and expressing the multimer havingdisulfide bond with hinge region of the antibody, or a method expressingchimera protein, in which antibody recognition region is expressed inthe C-terminal or N-terminal, and reacting with the polypeptidecontaining extracellular part of the thus said Delta-1 and HumanSerrate-1 and/the antibody which recognize specifically the antibodyrecognition region in the C-terminal or N-terminal. In the othermethods, a method, in which a fused protein expressed with only thehinge region of the antibody and the dimerized by disulfide bond, can bementioned. The multimer of human Delta-1 and human Serrate-1 havinghigher specific activity than the dimer can be obtained. The saidmultimer is constructed by fused protein which is prepared forexpressing the peptide in the C-terminal, N-terminal or other region.The protein is prepared in the form of forming disulfide bond withouteffecting in any activities of the other human Delta-1 or humanSerrate-1. The multimer structure can also be expressed by arranging oneor more peptide, which is selected from polypeptides containing aminoacids sequence of the sequence listing, SEQ ID NO: 2, 3, 5 or 6, withgenetic engineering method in series or in parallel. Other known methodsfor providing multimer structure having diner or higher can be applied.Accordingly, the present invention includes any polypeptides containingamino acid sequences described in the sequence listing, SEQ ID NO: 2, 3,5 or 6 in the form of dimer or higher more structure prepared by geneticengineering technique.

Further in the other method, multimerization method using chemicalcross-linker can be mentioned. For example, dimethylsuberimidatedihydrochloride for cross-linking lysine residue,N-(γ-maleimidebutyryloxy) succinimide for cross-linking thiol group ofcysteine residue and glutaraldehyde for cross-linking between aminogroups can be mentioned. The multimer with diner or more can besynthesized by applying these cross-linking reactions. Accordingly, thepresent invention includes any polypeptides containing amino acidsequences described in the sequence listing, SEQ ID NO: 2, 3, 5 or 6 inthe form of diner or more structure prepared by chemical cross-linkingagents.

In application of medical care in which cells are proliferated andactivated in vitro and are returned to the body, human Delta-1 or humanSerrate-1 of the form hereinabove can be added directly in the medium,but immobilization can also be made. Immobilization method includesapplying amino group or carboxyl group in the peptide, using suitablespacers or the above mentioned cross-linkers, and the polypeptide can becovalently bound to the culture vessels. Accordingly, the presentinvention includes any polypeptides containing amino acid sequencesdescribed in the sequence listing, SEQ ID NO: 2, 3, 5 or 6 in the formof existing on the solid surface.

Since the natural human Delta-1 and human Serrate-1 are cell membraneproteins, differentiation suppressive action in the Examples can beexpressed by cocultivating with cells expressing these molecules andblood undifferentiated cells. Consequently, this invention includescocultivation method with transformed cells by using DNA coding aminoacid sequences in the sequence listing, SEQ ID NO: 2–7 andundifferentiated cells.

Expressed cell may be COS-7 cell as shown in Examples, but cells ofhuman origin are preferable, and further expressed cells may be cellline or any of human in vivo blood cells and somatic cells.Consequently, the polypeptide can be expressed in vivo by integratedinto vectors for gene therapy.

As shown in Example 10, FLAG chimera protein of human Delta-1 or humanSerrate-1, both of which are low concentrated monomer, shows not acolony formation suppressive action but a colony formation stimulatingaction. This action may be involved in expressing Notch receptor andNotch ligand in the occasion of cell division of blood undifferentiatedcells and acting the polypeptide of the present invention as anantagonist for that action. This suggests that the polypeptide havingamino acid sequence of the sequence listing, SEQ ID NO: 1, 2, 4 or 5,shows colony formation stimulation action by controlling theconcentration of its action.

This fact suggests that inhibition of binding the polypeptide havingamino acid sequence in the sequence listing, SEQ ID NO: 2–7 and thesereceptors can be used for finding out molecules and compounds forstimulating cell differentiation. The methods include binding experimentusing radio isotope, luciferase assay using transcriptional controlfactors, a down stream molecule of the Notch receptor, and simulation onthe computer by X-ray structural analysis. Accordingly, the presentinvention includes screening method for pharmaceuticals usingpolypeptide in the sequence listing, SEQ ID NO: 2–7.

As shown in Example 13, specific leukemia cells can be differentiated byusing IgG chimera protein of human Delta-1 or human Serrate-1.Consequently, the present invention can be applied for diagnosticreagents for leukemia or isolation of specific blood cells. This resultindicates that human Delta-1 or human Serrate-1 molecule bindsspecifically with its receptor, a Notch receptor molecule. For example,expression of Notch receptor can be detected by using fused protein withthe above extracellular region and human IgGFc. Notch is known toinvolve in some type of leukemia (Ellisen et al., Cell 66, 649–661,1991). Accordingly, the polypeptide having amino acids sequence in thesequence listing. SEQ ID NO: 2, 3, 5 and 6 can be used for diagnosticreagents for in vitro or in vivo.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1: Alignment of DSL domain of Notch ligand identified in variousorganisms including the molecules of the present invention, wherein theconsensus sequence is SEQ ID NO: 40, hDelta-1.DSL is SEQ ID NO: 41,dDelta.DSL is SEQ ID NO: 42, xDelta.DSL is SEQ ID NO: 43, cDelta-1.DSLis SEQ ID NO: 44, mDelta-1.DSL is SEQ ID NO: 45, hSerrate-1.DSL is SEQID NO: 46, dSerrate.DSL is SEQ ID NO: 47, and rJagged.DSL is SEQ ID NO:48.

FIGS. 2A and 2B : Suppression of colony formation of the bloodundifferentiated cells using the molecules of the present invention.

FIG. 3: Concentration dependency of colony formation suppression of theblood undifferentiated cells using the molecules of the presentinvention.

FIG. 4: A graph showing calculation of LTC-1 after liquid culture usingthe molecules of the present invention.

FIGS. 5A and 5B: Cells stained by the molecules of the presentinvention.

EXAMPLES

Following examples illustrate the embodiments of the present inventionbut are not construed as limiting these examples.

Example 1

Cloning of PCR Products using Human Delta-1 Primer and Determination ofBase Sequence.

A mixed primer corresponding to amino acid sequence conserved inC-Delta-1 and X-Delta-1, i.e. sense primer DLTS1 (sequence listing, SEQID NO: 14) and antisense primer DLTA2 (sequence listing, SEQ ID NO: 15),were used.

A synthetic oligonucleotide was prepared by using automatic DNAsynthesizer with the principle of immobilized method. The automatic DNAsynthesizer used was 391PCR-MATE of Applied Biosystems Inc., U.S.A.Nucleotide, carrier immobilized with 3′-nucleotide, solution andreagents are used according to the instructions by the same corporation.Oligonucleotide was isolated from the carrier after finishing thedesignated coupling reaction and treating the oligonucleotide carrier,from which protective group of 5′-teminal was removed, with concentratedliquid ammonia at room temperature for one hour. For removing theprotective groups of nucleic acid and phosphoric acid, the reactantsolution containing nucleic acid was allowed to stand in theconcentrated ammonium solution in the sealed vial at 55° C. for over 14hours. Each oligonucleotide, from which the carrier and protectivegroups were removed, was purified by using OPC cartridge of the AppliedBiosystems Inc., and detritylated by using 2% trifluoracetic acid.Primer was dissolved in deionized water to set final concentration of100 pmol/μl after purification.

Amplification of these primers by PCR was performed as follows. Humanfetal brain originated cDNA mixed solution (QUICK-Clone cDNA, CLONTECHInc., U.S.A.) 1 μl was used. 10× buffer solution [500 mM KCl, 100 mMTris-HCl (pH 8.3), 15 mM MgCl₂, 0.01% gelatin] 5 μl, dNTP mixture(Takara Shuzo Co., Japan) 4 μl, sense primer DLTS1 (100 pmol/μl) 5 μlwhich was specific to the above vertebrates and antisense primer DLTA2(100 pmol/μl) 5 μl and TaqDNA polymerase (AmpliTaq, Takara Shuzo Co.,Japan, 5 U/μl) 0.2 μl were added thereto, and finally deionized waterwas added to set up total 50 μl, PCR was performed by 5 cycles of acycle consisting of treatment at 95° C. for 45 seconds, at 42° C. for 45seconds and 72° C. for 2 minutes, further 35 cycles of a cycleconsisting of treatment at 95° C. for 45 seconds, at 50° C. for 45seconds and 72° C. for 2 minutes, and finally allowed to stand at 72° C.for 7 minutes. A part of the PCR products was subjected to 2% agarosegel electrophoresis, stained with ethidium bromide (Nippon Gene Co.,Japan), and observed under ultraviolet light to confirm amplification ofabout 400 bp DNA.

Total amount of PCR product was subjected to electrophoresis with 2%agarose gel prepared by low melting point agarose (GIBCO BRL Inc.,U.S.A.), stained by ethidium bromide, cutting out about 400 bp bands ofPCR products by the Delta primer under the UV light, adding distilledwater of the same volume of the gel, heating at 65° C. for 10 minutes,and completely dissolving the gel. The dissolved gel was centrifuged at15000 rpm for 5 minutes to separate supernatant solution after addingequal volume of TE saturated phenol (Nippon Gene Co., Japan) and thesame separation operation was performed after adding TE saturatedphenol:chloroform (1:1) solution and chloroform. DNA was recovered fromthe final solution by ethanol precipitation.

A vector, pCRII vector (Invitorogen Inc., U.S.A., hereinafter designatesas pCRII) was used. The vector and the above DNA in molar ratio of 1:3were mixed and DNA was ligated into the vector by using T4 DNA ligase(Invitorogen Inc., U.S.A.). The pCRII, to which DNA was integrated, wassubjected to gene transduction into E. coli one shot competent cells(Invitorogen Inc., U.S.A.) and was spread on the semi-solid medium plateof L-Broth (Takara Shuzo Co., Japan) containing ampicillin (Sigma Corp.,U.S.A.) 50 μg/ml and allowed to stand at 37° C. for about 12 hours. Theresulting colonies were randomly selected, inoculated in the L-Brothliquid medium 2 ml containing same concentration of ampicillin and shakecultured at 37° C. for about 18 hours. The cultured bacterial cells wererecovered and the plasmid was separated by using Wizard Miniprep(Promega Inc., U.S.A.) according to the attached explanation sheet. Theplasmid was digested by restriction enzyme EcoRI. Integration of thesaid PCR product was confirmed by incision of about 400 bp DNA. Basesequence of the incorporated DNA in the confirmed clone was determinedby the fluorescent DNA sequencer (Model 373S, Applied System Inc.,U.S.A.)

Example 2

Cloning of Full Length Novel Human Delta-1 and its Analysis

A screening of clones having full length cDNA was performed byhybridization from human placenta origin cDNA library (inserted cDNA inλgt-11. CLONTECH Inc., U.S.A.) in plaques corresponding to 1×10⁶plaques. Generated plaques were transfered onto nylon filter (Hybond N+:Amersham Inc., U.S.A.). The transcribed nylon filter was subjected toalkaline treatment [allow to stand for 7 minutes on the filter paperpermeated with a mixture of 1.5 M NaCl and 0.5 M NaOH], followed by twoneutralizing treatments [allow to stand for 3 minutes on the filterpaper permeated with a mixture of 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2)and 1 mM EDTA]. Subsequently, the filter was shaken for 5 minutes in the2-fold concentrated SSPE solution [0.36 M NaCl, 0.02 M sodium phosphate(pH 7.7) and 2 mM EDTA], washed and air-dried. Then the filter wasallowed to stand for 20 minutes on the filter paper, which was permeatedwith 0.4 M NaOH, shaken for 5 minutes with 5-fold concentrated SSPEsolution and washed, then again air-dried. Screening was conducted inthe human Delta-1 probe labeled with radioisotope ³²P using thesefilters.

DNA probe prepared in Example 1 was labeled with ³²P as follows. A DNAfragment was cutted out by EcoRI from pCRII, inserted a purified PCRproduct (about 400 bp) by human Delta-1 primer and determined genesequence, and was isolated from low melting point agarose gel. The thusobtained DNA fragment was labeled by DNA labeling kit (Megaprime DNAlabeling system: Amersham, U.S.A.). The primer solution 5 μl anddeionized water were added to DNA 25 ng to set up total volume of 33 μl,which was treated for 5 minutes in boiling water bath. Reaction buffersolution 10 μl containing dNTP, α-³²P-dCTP 5 μl and T4 DNApolynucleotide kinase solution 2 μl were added thereto, treated at 37°C. for 10 minutes in water bath. Subsequently, the mixture was purifiedby Sephadex column (Quick Spin Column Sephadex G-50: Boehringer MannheimInc., Germany), then treated for 5 minutes in boiling water bath andice-cooled for 2 minutes for use.

Hybridization was performed as follows. The prepared filter hereinabovewas immersed into the prehybridization solution consisting of SSPEsolution, in which final concentration of each component is set at5-fold concentration, 5-fold concentration of Denhardt's solution (WakoPure Chemicals, Japan), 0.5% SDS (sodium dodecyl sulfate, Wako PureChemicals, Japan) and salmon sperm DNA (Sigma, U.S.A.) 10 μg/mldenatured by boiling water, and shaken at 65° C. for 2 hours, then thefilter was immersed into the hybridization solution of the samecomposition with the above prehybridization solution with the³²P-labeled probe above mentioned and shaken at 65° C. for 2 hours for16 hours to perform hybridization.

The filter was immersed into SSPE solution containing 0.1% SDS, shakenat 55° C. and washed twice, further immersed into 10-fold dilution ofSSPE solution containing 0.1% SDS and washed four times at 55° C. Anautoradiography of the washed filter was performed using intensifiedscreen. Clones of strongly exposed part were collected and the plaquesobtained were again spread and screened by the same method hereinbeforeto separate complete single clones.

The thus isolated phage clones were seven clones. Phage of all of theseclones was prepared to about 1×10⁹ pfu, purified the phage DNA, digestedby restriction enzyme EcoRI and inserted into pBluescript (StratageneInc., U.S.A.) which was digested EcoRI in the same way. DNA sequences ofthe both ends of these clones were analyzed by DNA sequencer. Threeclones of D5, D6 and D7 were the clone containing DNA sequence from No.1 to 2244 in the sequence listing, SEQ ID NO: 8. A clone D4 was a clonecontaining DNA sequence from No. 999 to 2663 in the sequence listing,SEQ ID NO: 8. The clones D5 and D4 prepared the deletion mutant by usingkilosequence deletion kit (Takara Shuzo Co., Japan) according to adescription of the attached paper. Full-length cDNA base sequence of thepresent invention was determined using the DNA sequencer from bothdirection of 5′-direction and 3′-direction.

By applying with XhoI site at No. 1214 in DNA sequence in the sequencelisting, SEQ ID NO: 8, D4 and D5 were digested by restriction enzymeXhoI to prepare plasmid pBSDel-1 containing full length of DNA sequencein the sequence listing, SEQ ID NO: 8.

Example 3

Cloning of Human Serrate-1 specific PCR Product and Determination ofBase Sequence

A mixed primer, which corresponded to amino acid sequence conserved inDrosophila Serrate and rat Jagged, i.e. sense primer SRTS 1 (thesequence listing, SEQ ID NO: 16) and antisense primer SRTA2 (thesequence listing, SEQ ID NO: 17), was used. Preparation was conducted bythe same way as described in Example 1.

Amplification by PCR using these primers was performed as follows. Tothe human fetal brain originated cDNA mixed solution hereinbefore 1 μlwas added 10× buffer solution (described in Example 1) 5 μl, said dNTPmixture 4 μl, sense primer SRTS1 (100 pmol/μl) 5 μl and antisense primerSRTA2 (100 pmol/μl) 5 μl specific to Serrate-1 homologue hereinbefore,and said TaqDNA polymerase 0.2 μl, and finally added deionized water toset up total volume 50 μl. The mixture was treated for 5 cycles of acycle consisting of at 95° C. for 45 seconds, at 42° C. for 45 secondsand 72° C. for 2 minutes, and 35 cycles of a cycle consisting of at 95°C. for 45 seconds, at 50° C. for 45 seconds and 72° C. for 2 minutes,and finally allowed to stand at 72° C. for 7 minutes to perform PCR. Apart of the PCR product was subjected to 2% agarose gel elctrophoresis,stained by ethidium bromide, and observed under ultraviolet light toconfirm amplification of about 500 bp cDNA.

Total amount of PCR product was subjected to electrophoresis with 2%agarose gel prepared by low melting point agarose, stained by ethidiumbromide, cutting out about 500 bp bands under the UV light, addingdistilled water of the equal volume of the gel, heating at 65° C. for 10minutes, and completely dissolving the gel. The dissolved gel wascentrifuged at 15000 rpm for 5 minutes to separate supernatant solutionafter adding equal volume of TE saturated phenol and the same separationoperation was performed after adding TE saturated phenol:chloroform(1:1) solution and chloroform. DNA was recovered from the final solutionby ethanol precipitation.

A vector, pCRII vector was used. The vector and the above DNA were mixedin molar ratio of 1:3 and DNA fragment was ligated into the vector pCRIIby the same method in Example 1. The pCRII, to which DNA was integrated,was subjected to gene transduction into E. coli. The resulting colonieswere randomly selected and were inoculated in liquid medium L-Broth 2 mlcontaining the same concentration of ampicillin and shake cultured at37° C. for about 18 hours. The cultured bacterial cells were recoveredand the plasmid was separated by using the Wizard Miniprep according tothe attached explanatory sheet. The plasmid was digested by restrictionenzyme EcoRI. Integration of the said PCR product was confirmed byincision of about 500 bp DNA. Base sequence of the incorporated DNA inthe confirmed clone was determined by the fluorescent DNA sequencer.

Example 4

Cloning of Full Length Novel Human Serrate-1 and its Analysis

A screening of clones having full length cDNA was performed byhybridization from the human placenta origin cDNA library hereinbeforein plaques corresponding to 1×10⁶ plaques. Preparation of the filter wasperformed by the same method as described in Example 2. Screening wasconducted in the human Serrate-1 probe labeled with radioisotope ³²Pusing the filter.

The above DNA probe labeled with ³²P was prepared by a method describedin Example 2, and hybridization, washing of the filter and isolation ofthe clone were performed by the description in Example 2.

The thus isolated phage clones were 22 clones. Phage of all of theseclones was prepared to about 1×10⁹ pfu, purified the phage DNA, digestedby restriction enzyme EcoRI and inserted into pBluescript which wasdigested EcoRI in the same way. DNA sequences of the both ends of theseclones were analyzed by DNA sequencer. Two clones of S16 and S20 werethe clone containing DNA sequence from No. 1 to 1873 in the sequencelisting, SEQ ID NO: 10. Two clones S5 and S14 were the clones containingDNA sequence from No. 990 to 4005 in the sequence listing, SEQ ID NO:10. These clones prepared the deletion mutants by using the kilosequencedeletion kit according to a description of the attached leaflet. ThecDNA base sequence coding the polypeptide of the present invention wasdetermined using the DNA sequencer from both direction of 5′-directionand 3′-direction.

By applying with BglII site at No. 1293 in DNA sequence in the sequencelisting, SEQ ID NO: 10.S20 and S5 were digested by restriction enzymeBglII, and DNA of gene sequence from No. 1 to 4005 in the sequencelisting SEQ ID NO: 10 was subcloned in E.coli vector pBluescript. Thisplasmid is named as pBSSRT.

Since the termination codon was not found in the C-terminal and theintracellular region coding C-terminal amino acids was not cloned,cloning of the full length gene was performed using the 3′ RACE systemkit, GIBCO-BRL, U.S.A., according to the description of the attachedleaflet. The cloning of cDNA gene for 3′-direction was performed inpolyA⁺ RNA (CLONTECH Inc., U.S.A.) originated from human placenta todetermine the gene sequence.

The thus cloned three gene fragments by applying with BglII site in DNAsequence No. 1293 and AccI site in DNA sequence No. 3943 and a plasmidcontaining full length of DNA sequence in the sequence listing, SEQ IDNO: 5 were inserted between EcoRI and Xbal in the multi-cloning site ofpUC18 to prepare pUCSR-1 containing full length gene of human Serrate-1.This gene sequence as well as its amino acid sequence is shown in thesequence listing, SEQ ID NO: 10.

Example 5

Preparation of Expression Vectors of Human Delta-1

Using the gene consisting of DNA sequence described in the sequencelisting, SEQ ID NO: 7, expression vectors of human Delta-1 proteinmentioned in the following 1)–5) were prepared. Addition of restrictionenzyme sites and insertion of short gene sequence were performed usingExSite PCR-Based Site-Directed Mutagenesis Kit (Stratagene Inc., U.S.A.)according to the operating manual.

1) Expression Vector of Soluble Human Delta-1 Protein (HDEX)

The cDNA coding polypeptide of amino acid sequence form No. 1 to 520 inthe sequence listing, SEQ ID NO: 3 was ligated with expression vectorpMKITNeo containing SRα promoter and neomycin resistance gene to prepareexpression vector.

For preparation of expression vector of human Delta-1, in order tostable expression from gene product, EcoRI site was added in the 20 bpupper stream for 5′-direction of the initiation codon (gene sequence No.179 in the sequence listing, SEQ ID NO: 8). Using the above MutagenesisKit, a plasmid pBSDel-1, which contained DNA sequence in sequencelisting, SEQ ID NO: 8 and full length cDNA of human Delta-1 were set asthe template, and oligonucleotides having gene sequence in sequencelisting, SEQ ID NO: 18 and SEQ ID NO: 19 was set as the primers. ThenDNA adding EcoRI site in the 20 bp upper stream for 5′-direction wasprepared. Hereinafter this plasmid is designated as pBS/Eco-Delta.

The pBS/Eco-Delta was used as a template. In order to add thetermination codon and restriction enzyme MluI site after a C-terminalposition, using the Mutagenesis Kit, and setting oligonucleotides havinggene sequences in the sequence listing, SEQ ID NO: 20 and SEQ ID NO: 21as primers, addition of the termination codon and MluI site wereperformed. The resulted vector was digested by EcoRI and MluI, and about1600 bp splitted gene fragment was ligated in pMKITNeo, which wastreated by the same restriction enzyme, to construct the expressionvector. This vector was designated as pHDEX.

2) Expression Vector of FLAG Chimera Protein of Soluble Human Delta-1(HDEXFLAG)

The cDNA coding chimera protein, to which cDNA coding FLAG sequence wasadded to the C-terminal of polypeptide from No. 1 to 520 of amino acidsequence in the sequence listing, SEQ ID NO: 3, was ligated to theexpression vector pMKITNeo containing SRα promoter and neomycinresistance gene to prepare the expression vector.

Using pBS/Eco-Delta as template, FLAG sequence was added in theextracellular C-terminal, i.e. after Gly at No. 520 in the sequencelisting, SEQ ID NO: 3. In order to add the termination condon andrestriction enzyme MluI site, using the Mutagenesis Kit, and settingoligonucleotides having gene sequence in the sequence listing, SEQ IDNO: 22 and SEQ ID NO: 21 as primers, a gene coding FLAG sequence andtermination codon and MluI site were added in the C-terminal. Thisvector was digested by EcoRI and MluI, and about 1700 bp splitted genefragment was ligated to the similarly restriction enzyme treatedpMKITNeo to construct the expression vector. This vector was designatedas pHDEXFLAG.

3) Expression Vector of IgG1Fc Chimera Protein of Soluble Human Delta-1(HDEXIg)

A gene sequence coding polypeptide, to which amino acid sequence of Fcregion below the hinge part of human IgG1 was added to the C-terminal ofpolypeptide having amino acid sequence in the sequence listing, SEQ IDNO: 3.

Preparation of fused protein with immunoglobulin Fc protein wasperformed according to the method of Zettlmeissl et al. (Zettlmeissl etal., DNA cell Biol., 9, 347–354, 1990). A gene using genome DNA withintron was applied and the said gene was prepared by using PCR. Humangenome was used as a template. An oligonucleotide of the sequence in thesequence listing, SEQ ID NO: 23 with restriction enzyme BamHI site andan oligonucleotide of the sequence in the sequence listing, SEQ ID NO:24 with restriction enzyme XbaI site were used as primers. PCR wasperformed using the primers and human genomic DNA as template. About 1.4kbp band was purified, treated by restriction enzyme BamHI and XbaI(Takara Shuzo Co., Japan), and genes were ligated to pBluescript, whichwas similarly treated by restriction enzyme, by using T4 DNA ligase toprepare subcloning. Later, the plasmid DNA was purified and sequenced toconfirm gene sequence, then the said gene sequence was confirmed asgenomic DNA in the hinge region of heavy chain of the human IgG1. (Thesequence is referred to Kabat et al., Sequence of ImmunologicalInterest, NIH Publication No. 91-3242, 1991). Hereinafter this plasmidis designated as pBShIgFc.

Using the said pBS/Eco-Delta as template, and using the Mutagenesis Kit,restriction enzyme BamHI site was added in the extracellular C-terminal,i.e. after Gly at No. 520 in the sequence listing, SEQ ID NO: 3.Furthermore, in order to add restriction enzyme XbaI and MluI sites tothe downstream, and setting the oligonucleotides having gene sequence inthe sequence listing, SEQ ID NO: 25 and SEQ ID NO: 26. using theMutagenesis Kit, BamHI, XbaI and MluI sites were added. This vectordigested by XbaI and BamHI and about 1200 bp of gene fragment digestedfrom the above pBShIgFc by XbaI and BamHI were ligated to prepare vectorcontaining gene fragments coding the final objective soluble humanDelta-1 IgG1Fc chimera protein. Finally, this vector was digested byEcoRI and MluI and about 3000 bp splitted gene fragments were ligatedwith the similarly restriction enzyme treated pMKITNeo to construct theexpression vector. This vector was designated as pHDEXIg.

4) Expression Vector of Full Length Human Delta-1 Protein (HDF)

The cDNA coding polypeptide from No. 1 to 702 of amino acid sequence inthe sequence listing, SEQ ID NO: 4, was ligated to the expression vectorpMKITNeo containing SRα promoter and neomycin resistance gene to preparethe expression vector.

In order to add the termination codon in C-terminal of the full lengthsequence, i.e. after Val at No. 702 in the sequence listing, SEQ ID NO:4 and restriction enzyme MluI site, using the Mutagenesis Kit andpBS/Eco-Delta as template and setting oligonucleotides having genesequence in the sequence listing, SEQ ID NO: 27 and SEQ ID NO: 28 asprimers, the termination codon and MluI site were added in theC-terminal. This vector was digested by EcoRI and MluI, and about 2200bp splitted gene fragment was ligated to the similar restriction enzymetreated pMKITNeo to construct the expression vector. This vector wasdesignated as pHDF.

5) Expression Vector of FLAG Chimera Protein (HDFLAG) of Full LengthHuman Delta-1

The cDNA coding chimera protein, to which cDNA coding FLAG sequence wasadded to the C-terminal of polypeptide from No. 1 to 702 of amino acidsequence in the sequence listing, SEQ ID NO: 4, was ligated to theexpression vector pMKITNeo containing SRα promoter and neomycinresistance gene to prepare the expression vector.

In order to add FLAG sequence in the C-terminal, the termination codonand restriction enzyme MluI site, setting oligonucleotides having genesequence in the sequence listing, SEQ ID NO: 29 and SEQ ID NO: 28 asprimers and using pBS/Eco-Delta as template, a gene coding FLAG sequenceand termination codon and MluI site were added in the C-terminal. Fromthis vector, DNA coding full length of human Delta-1 was cloned in E.coli vector pUC19 to prepare vector pUCDL-1F coding full length of humanDelta-1. This vector was digested by EcoRI and MluI, and about 2200 bpsplitted gene fragments were ligated to the similar restriction enzymetreated pMKITNeo to construct the expression vector. This vector wasdesignated as pHDFLAG.

Example 6

Preparation of Expression Vectors of Human Serrate-1

Using the gene consisting of DNA sequence described in the sequencelisting, SEQ ID NO: 10 expression vectors of human Serrate-1 proteinmentioned in the following 6)–10) were prepared. Addition of restrictionenzyme sites and insertion of short gene sequence were performed byusing the ExSite PCR-Based Site-Directed Mutagenesis Kit as well asaccording to the operating manual.

6) Expression Vector of Soluble Human Serrate-1 Protein (HSEX)

The cDNA coding polypeptide of amino acid sequence form No. 1 to 1036 inthe sequence listing, SEQ ID NO: 6 was ligated with expression vectorpMKITNeo to prepare expression vector.

For preparation of expression vector of polypeptide expression cellshaving amino acid sequence from No. 1 to 1036 in the sequence listing,SEQ ID NO: 6, in order to express gene product more stably EcoRI sitewas added in the 10 bp upper stream region for 5′-direction of theinitiation codon (gene sequence No. 409 in the sequence listing, SEQ IDNO; 10). Using the above Mutagenesis Kit, a plasmid pBSSRT, whichcontained cDNA of human Serrate-1 from No. 1 to 4005 of DNA sequence inthe sequence listing. SEQ ID NO: 10, was set as the template, andoligonucleotide having gene sequence in sequence listing, SEQ ID NO: 30and oligonucleotide having gene sequence in sequence listing, SEQ ID NO:31 were set as the primers. Then DNA adding EcoRI site in the 10 bpupper stream for 5′-direction was prepared.

The thus prepared vector (hereinafter designates as pBS/Eco-Serrate-1)was used as a template. In order to add the termination codon andfurther restriction enzyme MluI site in the extracellular C-terminalregion, i.e. C-terminal of polypeptide in the sequence listing, SEQ IDNO: 6, using the Mutagenesis Kit, and setting oligonucleotide havinggene sequence in the sequence listing, SEQ ID NO: 32 and oligonucleotidehaving gene sequence in the sequence listing, SEQ ID NO: 33, as primers,the termination codon and MluI site were added. The resulting vector wasdigested by EcoRI and MluI, and about 3200 bp splitted gene fragment wasligated in pMKITNeo, which was treated by the same restriction enzyme,to construct the expression vector. This vector was designated as pHSEX.

7) Expression Vector of FLAG Chimera Protein of Soluble Human Serrate-1(HSEXFLAG)

The cDNA coding FLAG chimera protein, which had FLAG sequence in theC-terminal of polypeptide from No. 1 to 1036 of amino acid sequence inthe sequence listing, SEQ ID NO: 6, was ligated to the expression vectorpMKITNeo containing SRα promoter and neomycin resistance gene to preparethe expression vector.

Using pBS/Eco-Serrate-1 as a template, FLAG sequence was added in theextracellular C-terminal, i.e. the C-terminal of polypeptide in thesequence listing, SEQ ID NO: 6. In order to add the termination codonand further restriction enzyme MluI site, using the Mutagenesis Kit, andsetting oligonucleotide having gene sequence in the sequence listing,SEQ ID NO: 34 and oligonucleotide having gene sequence in the sequencelisting, SEQ ID NO: 33 as primers, a gene coding FLAG sequence andtermination codon and MluI site were added in the C-terminal. Thisvector was digested by EcoRI and MluI, and about 3200 bp splitted genefragment was ligated to the similarly restriction enzyme treatedpMKITNeo to construct the exprssion vector. This vector was designatedas pHSEXFLAG.

8) Expression Vector of IgG1Fc Chimera Protein of Soluble HumanSerrate-1 (HSEXIg)

A gene sequence coding polypeptide, to which amino acid sequence of Fcregion below the hinge part of human IgG1 was added to the C-terminal ofpolypeptide having amino acid sequence in the sequence listing, SEQ IDNO: 6.

In order to add restriction enzyme BamHI site in the extracellularC-terminal, i.e. after the polypeptide having the sequence in thesequence listing, SEQ ID NO: 6 and further restriction enzyme XbaI andMluI sites to its downstream, BamHI, XbaI and MluI sites were addedUsing pBS/Eco-Serrate-1 as a template by the Mutagenesis Kit, usingoligonucleotide having gene sequence in the sequence listing, SEQ ID NO:35 and oligonucleotide having gene sequence in the sequence listing, SEQID NO: 36 as primers. This vector digested by XbaI and BamHI and about1200 bp of gene fragment digested from the above pBShIgFc by XbaI andBamHI were ligated to finally prepare a vector, which contained genefragments coding IgG1Fc chimera protein of the soluble human Serrate-1.Finally, this vector was digested by EcoRI and MluI, and splitted about4400 bp gene fragment was ligated to pMKITNeo to construct theexpression vector. This vector was designated as pHSEXIg.

9) Expression Vector of Full Length Human Serrate-1 Protein (HSF)

The cDNA coding polypeptide from No. 1 to 1187 of amino acid sequence inthe sequence listing, SEQ ID NO: 7 was ligated with expression vectorpMKITNeo containing SRα promoter and neomycin resistance gene to prepareexpression vector.

For preparation of the full length expression vector about 900 bpsplitted gene fragment from pBS/Eco-Serrate-1 digested by restrictionenzyme EcoRI and BglII, and pUCSR-1 digested by the same restrictionenzyme were ligated, and a vector pUC/Eco-Serrate-1 coding full lengthgene of human Serrate-1 was prepared.

In order to add the termination codon to the site after Val at No. 1187in the sequence listing, SEQ ID NO: 7, and further add the restrictionenzyme MluI site, using the Mutagenesis Kit, the termination codon andMluI site were added to the C-terminal using oligonucleotides havinggene sequence in the sequence listing, SEQ ID NO: 37 and SEQ ID NO: 38as primers and the pBS/Eco-Serrate-1as a template. The resulting vectorwas digested by EcoRI and MulI, and about 3700 bp splitted genefragments were ligated in pMKITNeo, which was treated by the samerestriction enzyme, to construct the expression vector. This vector wasdesignated as pHSF.

10) Expression Vector of FLAG Chimera Protein of Full Length HumanSerrate-1 (HSFLAG)

The cDNA coding chimera protein, to which cDNA coding FLAG sequence wasadded in the C-terminal of polypeptide from No. 1 to 1187 of amino acidsequence in the sequence listing, SEQ ID NO: 7, was ligated to theexpression vector pMKITNeo containing SRα promoter and neomycinresistance gene to prepare the expression vector.

In order to add FLAG sequence in the C-terminal the termination codonand further restriction enzyme MluI site, setting oligonucleotideshaving gene sequence in the sequence listing, SEQ ID NO: 39 and SEQ IDNO: 38 as primers, using pBS/Eco-Serrate-1 as a template a gene codingFLAG sequence, the termination codon and the MluI site were added in theC-terminal as same as similar manner. This vector was digested by EcoRIand MluI, and about 3700 bp splitted gene fragments were ligated to thesimilarly restriction enzyme treated pMKITNeo to construct theexpression vector. This vector was designated as pHSFLAG.

Example 7

Expression and Gene Transfer of the Expression Vectors into Cells

The expression vectors prepared in Examples 5 and 6 were transduced intoCOS-7 cell (obtained from RIKEN Cell Bank, Physical and ChemicalResearch Institute, Japan, RCB0539).

Cell culture before gene transduction was performed by culturing inD-MEM (Dulbecco modified Eagle's medium, GIBCO-BRL Inc., U.S.A.) 10%FCS. On a day before gene transduction, medium of cells was changed toset cell counts 5×10⁵ cells/ml and cultured for overnight. On the day ofgene transduction, cells were sedimented by centrifugation,centrifugally washed twice with PBS(−) and prepared the cells to 1×10⁷cells/ml in 1 mM MgCl₂ and PBS(−). Gene transfer was performed byelectroporation using gene transduction device Gene-pulsar (Bio-RadInc., U.S.A.). The above cell suspension 500 μl was collected in thecell for electroporation (0.4 cm), added expression vector 20 μg, andallowed to stand in ice for 5 minutes. Electroporation was performedunder the condition 3 μF, 450 V twice, during the twice electroporationcell mixture was allowed to stand at room temperature. After 5 minutesstayed in ice, cells were spread in the culture medium, diameter 10 cmpreviously added 10 ml of medium, and cultured at 37° C. in 5% carbondioxide incubator.

The next day, the culture supernatant solution was removed, washed thecells adhered to the dish twice with PBS(−) 10 ml. In case of expressionvector pHDEX, pHDEXFLAG, pHDEXIg, pHSEX, pHSEXFLAG, and pHSEXIg,serum-free D-MEM 10 ml was added and cultured for 7 days. Culturesupernatant solution was recovered and was replaced the buffer to PBS(−)by Centricon 30 (Amicon Inc., U.S.A.) and simultaneously the solutionwas concentrated to 10-fold to obtain cell culture supernatant solution.

In case of pHDF, pHDFLAG, pHSF, and pHSFLAG, medium was changed by D-MEMcontaining 10% FCS. and cultured-further 3 days to prepare cell lysate.Thus, 2×10⁶ cells were suspended in the cell lysis buffer [50 mM Hepes(pH 7.5), 1% Triton X100, 10% glycerol, 4 mM EDTA, 50 μg/ml Aprotinin,100 μM Leupeptin, 25 μM Pepstatin A and 1 mM PMISF] 200 μl, allowed tostand in ice for 20 minutes and centrifuged at 14000 rpm for 20 minutesto remove supernatant solution to obtain cell lysate.

Expression of Proteins were Detected by Western Blotting.

Concentrated cultured supernatants or cell lysates were subjected toSDS-PAGE using an electrophoresis tank and polyacrylamide gel forSDS-PAGE (gradient gel 5–15%) (ACI Japan Inc., Japan) according to themanufacturer's construction. Samples were prepared by treatment inboiling water for 5 min. with 2-mercaptoethanol (2-ME) for reduction,and non-reduced condition without taking the above treatment. As amarker, Rainbow darker (high molecular weight, Amersham Inc.) was used.Sample buffer solution and electrophoresis buffer were prepared withreference to the attached leaflet. When the SDS-PAGE was finished,acrylamide gel was transcribed to PVDF membrane filter (BioRad Inc.,U.S.A.) using the Mini Trans Blot Cell (BioRad Inc.).

The thus prepared filter was shaken overnight at 4° C. in the Blockace(Dainippon Pharm. Co., Japan), TBS-T [20 mM Tris, 137 mM NaCl (pH 7.6)and 0.1% Tween 20] to blocking. According to the explanation of theattached leaflet of ECL Western blotting detection system (AmershamInc., U.S.A.): in case that the objective protein was human Delta-1origin, anti-human Delta-1 mouse monoclonal antibody described inExample 9 was used as primary antibody; in case that protein was humanSerrate-1 origin, anti-human Serrate-1 mouse monoclonal antibodydescribed in Example 9 was used as primary antibody; and in case thatprotein was FLAG chimera, anti-FLAG M2 mouse monoclonal antibody(Eastman Kodak, U.S.A.) was used as primary antibody, and peroxidaselabeled anti-mouse Ig sheep antibodies (Amersham Inc., U.S.A.) wasreacted. In case of IgG chimera, peroxidase labeled anti-human Ig sheepantibodies (Amersham Inc., U.S.A.) Was reacted.

Reaction time for antibodies was 1 hour at room temperature, and at aninterval of each reaction, washing was performed by shaking in TBS-T atroom temperature for 10 minutes for three times. After the finalwashing, the filter was immersed in the reaction solution of ECL-Westernblotting detection system (Amersham Inc., U.S.A.) for 5 minutes, andwrapped in polyvinylidene chloride wrap to expose X-ray film.

As the result, in the sample with treatment of reduction, the bandsshowing protein obtained by transduction of pHDEX and pHDEXFLAG wasdetetcd about 65 kD; protein obtained by transduction of pHDEXIg wasdetected about 95 kD, and protein obtained by transduction of pHDF andpHDFLAG was detected about 85 kD. In the non-reduced sample, the bandsshowing protein obtained by transduction of pHDEXIg was detectedslightly smeared bands at 120 kD to 200 kD, mainly about 180 kD, whichshowed about 2-fold of the reduction stage, consequently, dimer wasformed.

And also, in the sample with treatment of reduction, the bands showingprotein obtained by transduction of pHSEX and pHSEXFLAG was detectedabout 140 kD; protein obtained by transduction of pHSEXIg was detectedabout 170 kD, and protein obtained by transduction of pHSF and pHSFLAGwas detected about 150 kD. In the non-reduced sample, the bands showingprotein obtained by transduction of pHSEXIg was detected slightlysmeared bands at 250 kD to 400 kD, mainly about 300 kD, which showedabout 2-fold of the reduction stage, consequently, dimer was formed.

In these experiments, however cell lysate and cultured supernatant ofCOS-7 cells, to which pMKITNeo vector was transduced as a control wastested., no bands reacted against anti-human Delta-1 mouse monoclonalantibody, anti-human Serrate-1 mouse monoclonal antibody, anti-FLAGantibody, and anti-human Ig antibody were detected.

Therefore, this ten-expression vector can produce the objectivepolypeptides.

Example 8

Purification of Soluble Human Delta-1 and Human Serrate-1 Proteins ofGene Transduction Cells

Cultured supernatant of COS-7 cells consisting of HDBXFLAG, HDBXIg,HSEXFLAG and HSEXIg, all of which expression was detected by a method inExample 7, were prepared on large scale, and each chimera protein waspurified by affinity column chromatography.

In case of HDEXFLAG and HSEXFLAG, 2 liter of the cultured supernatantobtained by the method in Example 7 was passed through a column packedwith Anti-FLAG M2 Affinity Gel (Eastman Kodak, U.S.A.). The chimeraprotein was adsorbed in a column by a reaction of affinity of anti-FLAGantibody of the gel and FLAG sequence of the chimera protein. Column,inner diameter 10 mm, disposable column (BioRad Inc., U.S.A.) was usedwith packing the above gel 5 ml. A circulation system consisting ofmedium bottle→column→peristaltic pump→medium bottle was set up. Thecirculation was run by a flow 1 ml/min. for 72 hours. Thereafter thecolumn was washed with PBS (−) 35 ml and eluted by 0.5 M Tris-glycine(pH 3.0) 50 ml. The eluate of 25 fractions, each 2 ml, was collectedinto the tube, and each fraction was neutralized by 200 μl of 0.5 MTris-HCl (pH 9.5) previously added in each tube.

The eluate fraction, each 10 μl of the secretor FLAG chimera proteinwhich was purified by the above method was subjected to reductiontreatment described in Example 7. SDS-PAGE electrophoresis by 5–10%gradient polyacrylamide gel was performed. After finishing theelectrophoresis, silver staining was conducted by using Wako silverstain kit It (Wako Pure Chemicals, Japan) according to the explanationof the attached leaflet. Fractions from No. 4 to 8 showed detectablebands in HSFLAG. The size is identical with the result of Westernblotting of anti-FLAG antibody obtained in Example 6 in both of HDEXFLAGand HSEXFLAG. Therefore, purified HDEXFLAG and HSEFLAG were obtained.

In the IgG1Fc chimera protein, i.e. HDEXIg and HSEXIg, the culturedsupernatant solution 2 liter was adsorbed in Protein A Sepharose colulnn(Pharmacia Inc., Sweden) according to the same method as of FLAG chimeraprotein to collect the eluate fractions. Using a part of eluate as sameas in FLAG chimera protein, a determination of the eluate fraction,identification of the size and detection of the purity were performed bySDS-PAGE electrophoresis and silver staining in the reduced condition.Therefore, the eluate fraction from No. 4 to 15 were the detected bands.The size thereof is identical with the result of Western blotting usinganti-human Ig antibody in both of HDEXIg and HSEXIg. Therefore, purifiedHDEXIg and HSEXIg were obtained.

Example 9

Preparation of Antibodies Recognizing Human Delta-1 and Human Serrate-1

HDEXFLAG and HSEXFLAG, purified by the method in Example 8, were used asimmunogen, and rabbits were immunized. After assaying antibody titer,whole blood was collected and serum was obtained. Anti-human Delta-1rabbit polyclonal antibody and anti-human Serrate-1 rabbit polyclonalantibody were purified by using the econopack serum lgG purification kit(BioRad Inc., U.S.A.) with reference to the attached explanationleaflet.

HDEXFLAG and HSEXFLAG purified by a method described in Example 8 wereused as lmmunogens, and mouse monoclonal antibodies were preparedaccording to the explanation of the textbook. The purified HDEXFLAG orHSEXFLAG was administered in Balb/c mice (Nippon SLC CO., Japan)separately, 10 μg/mouse, immunized intracutaneously and subcutaneously.After second immunization increased serum titer was confirmed bycollecting blood ophthalmologically, the third immunization wasperformed. Subsequently, the spleen of mice was collected and fused withmouse myeloma cells P3×63Ag8 (ATCC TIB9) using polyethylene glycol.Hybridoma was selected by HAT medium (Immunological and BiologicalResearch Institute, Japan), and the hybridoma strains which producedantibody specifically recognizing extracellular region of human Delta-1or human Serrate-1 in the medium, were isolated by enzyme immunoassay.The hybridoma strains producing mouse monoclonal antibody, whichspecifically recognized human Delta-1 or human Serrate-1, wereestablished.

Anti-human Delta-1 monoclonal antibody and anti-human Serrate-1monoclonal antibody were purified and prepared by using Mab Trap GII(Pharmacia Inc., Sweden) and according to the explanation of theleaflet, from the supernatant of the thus established hybridoma.

Affinity column was prepared by using these monoclonal antibodies.Preparation of the affinity column was performed according to theexplanation attached to the CNBr activated Sephadex 4B (Pharmacia Inc.,Sweden). A column, 2 cm²×1 cm, containing gel 2 ml, was prepared.

A concentrated solution of the supernatant of the cultured COS-7 cells,to which pHDEX was gene transduced, was passed through the column forwhich anti-human Delta-1 monoclonal antibody was bound. A concentratedsolution of the supernatant of the cultured COS-7 cells, to which pHSEXwas gene transduced, was passed through the column, for which anti-humanSerrate-1 monoclonal antibody was bound. Each supernatant solution waspassed at 20 ml/hr, subsequently PBS (−) 15 ml was passed at the sameflow rate and washed the column. Finally, the products were eluted by amixture of 0.1 M sodium acetate and 0.5 M NaCl (pH 4.0). The eluate,each 1 ml fraction, was collected, and was neutralized by adding 1MTris-HCl (pH 9.1) 200 μl for each fraction.

SDS-PAGE of each purified protein was conducted under reduced conditionaccording to the method described in Example 8, followed by silverstaining and Western blotting to estimate molecular weight. HDEX, about65 kD, was purified from concentrated supernatant of the cultured COS-7cells, to which pHDEX was gene transduced, and HDSEX, about 140 kD, waspurified from concentrated supernatant of the cultured COS-7 cells, towhich pHSEX was gene transduced. Consequently, human Delta-1 and humanSerrate-1 can be purified by these affinity columns.

Example 10

Effects of HDEXIg and HSEXIg to Colony Formation of BloodUndifferentiated Cells

In order to observe physiological action of HDEXIg and HSEXIg on bloodundifferentiated cells, CD34 positive cells were cultured in theserum-free semi solid medium in the presence of HDEXIg and HSEXIg andknown cytokines, and number of colony forming cells were observed.

Human umbilical cord blood or adult human normal bone marrow blood wastreated by the silica solution (immunological and Biological ResearchInstitute. Japan) according to the attached explanation leaflet.Thereafter the low density cellular fraction (<1.077 g/ml) wasfractionated by densitometric centrifugation of Ficoll pack (PharmaciaInc., Sweden) to prepare mononuclear cells. CD34 positive cells of humanumbilical cord blood or human normal bone marrow blood was isolated fromthe mononuclear cells.

Separation of CD34 positive cells was performed by using Micro-SelectorSystem (AIS Inc., U.S.A.) or Dynabeads M-450 CD34 and DETACHa-BEADS CD34(Dynal Inc., Norway) according to attached explanation leaflets. Afterseparation, the purity was measured as follows. Cells were stained byFITC labeled CD34 antibody HPCA2 (Beckton-Deckinson Inc., U.S.A.) andexamined by a flow-cytometer (FACSCalibur, Beckton-Deckinson. U.S.A.).Purity above 85% was confirmed for use.

The thus isolated CD34 positive cells were suspended homogeneously toform 400 cells/ml of the medium hereinbelow, and spread in the 35 mmdish (Falcon Inc., U.S.A.), then cultured for 2 weeks in carbon dioxideincubator at 37° C. under 5% carbon dioxide, 5% oxygen, 90% nitrogen and100% humidity. The formed blood colonies were counted under the invertmicroscope.

A medium used is α-medium (GIBCO-BRL, U.S.A.), containing 2% deionizedbovine serum albumin (BSA. Sigma, U.S.A.). 10 μg/ml human insulin(Sigma, U.S.A.) 200 μg/ml transferrin (Sigma, U.S.A.), 10⁻⁵M2-mercaptoethanol (Nakarai Tesk Co., Japan), 160 μg/ml soybean lectin(Sigma. U.S.A.). 96 μg/ml cholesterol (Sigma, U.S.A. ) and 0.9%methylcellulose (Wako Pure Chemicals, Japan).

To the above medium under the following three conditions of cytokines,human Delta-1 extracellular Ig chimera protein (HDEXIg) or humanSerrate-1 extracellular Ig chimera protein (HSEXIg) were added to thefinal concentration of 1 μg/ml. For control, human IgG1 (Ahens Researchand Technology Inc., U.S.A.) was added with the same concentration inorder to observe effect of IgGFc region.

Conditions of cytokines are as follows.

-   1:100 μg/ml, human SCF(Intergen Inc., U.S.A.), 10 ng/ml humnan IL-3    (Intergen Inc., U.S.A.), 100 ng/ml human IL-6 (Intergen Inc.,    U.S.A.)-   2:100 ng/ml human SCF, 10 ng/ml human IL-3, 4 ng/ml human    thrombopoietin (Pepro Tech Inc., U.S.A.)-   3:100 ng/ml human SCF, 10 ng/ml human IL-3, 100 ng/ml human IL-6, 2    U/ml Epo (Chugai Seiyaku Co., Japan) 10 ng/ml human G-CSF (Chugai    Seiyaku Co., Japan)

Results are shown in FIG. 2. In FIG. 2, A is a case of human Delta-1extracellular Ig chimera protein (HDEXIg), and B is a case of humanSerrate-1 extracellular Ig chimera protein (HSEXIg). For A and B, eachdifferent origin human umbilical cord blood CD34 positive cell was used.The vertical axis: number of colonies. White: control, black: HDEXIg orHSEXIg. Both HDEXIg and HSEXIg have suppressive action of colonyformation. No differences of the activities on the types of colonieswere noted. Therefore, the molecular of the present invention hassuppressive action for colony formation against colony forming cells ofblood undifferentiated cells, i.e. diferentiation-suppressive action.Comparison with or without SCF on the activity indicated that thesuppressive action tended to occur only in the presence of SCF.

Dose-dependent manner of the activity was studied. Comparison with dimerHSEXIg and monomer HSEXFLAG was performed. Result is shown in FIG. 3.Concentration in this case is indicated as molar concentration. Forcomparison with dimer and monomer, dimer HSEXIg was indicated by exacttwo molar concentrations, and was plotted equivalent molar concentrationof the human Serrate-1. Vertical axis indicates colony forming countsand horizontal axis indicates molar concentration. Colony forming countswithout Notch ligand were plotted on the vertical axis in the zeroconcentration. For comparison, colony forming counts of human IgG1 1μg/ml, was about 100 colonies.

This result indicated that HSEXIg and HSEXFLAG suppressed colonyformation in dose-dependent manner. Activity of dimer HSEXIg wasstronger than the monomer. A monomer HSEXFLAG showed stimulative actionfor colony formation in the low concentration area.

Example 11

Actions of HDEXIg and HSEXIg on Long Term Liquid Culture of ColonyForming Blood Undifferentiated Cells

For observing physiological action of HDEXIg and HSEXIg on the bloodundifferentiated cells, umbilical cord blood CD34 positive cells wereculture for long term in the serum-free liquid medium in the presence ofHDEXIg or HSEXIg and known cytokines, and numbers of colony formingcells were observed.

The umbilical cord blood mononuclear CD34 positive cells separated by amethod described in Example 10 were liquid cultured at 1000 cells/wellin the 24 well cell culture plate (Falcon Inc., U.S.A.). Culture wasperformed at 37° C. in the carbon dioxide incubator under 5% carbondioxide and 100% humidity. Liquid culture medium was Iscove's modifiedDulbecco's medium (IMDM, GIBCO-BRL, U.S.A.) added with 2% BSA, 10 μg/mlhuman insulin, 200 μg/ml transferrin, 40 μg/ml low density lipoprotein(GIBCO-BRL, U.S.A.), 10⁻⁵M 2-mercaptoethanol, 50 ng/ml human SCF, 5ng/ml human IL-3, 10 ng/ml human IL-6, 5 ng/ml human GM-CSF (IntergenInc., U.S.A.), and 3 U/ml Epo. If necessary, XDEXIg-500 ng HSEXIg or 50ng/ml MIP-1α (Intergen Inc., U.S.A.) was added. The medium was added 1ml/well and half of the medium was changed three times in a week. Afterculturing 2, 4, 6 and 8 weeks, all cells were collected from wells byusing cell scraper in 1.5 ml micro tube. Cells were precipitated bycentrifugation and resuspended in a fresh IMDM 1 ml, counted the cellcounts by using hemocytometer, and in 5000 cells/ml, blood cell colonyforming assay was performed.

Blood cell colony forming assay was performed using the Iscove'smethylcellulose complete ready mix (Stem Cell Technologies Inc.,Canada), and each 1 ml was inoculated in two plates of 35 mm dish(Falcon Inc., U.S.A.) and incubated for 2 weeks in the carbon dioxideincubator. Blood colonies were counted CFU-GM and BFU-E in the invertmicroscope, and total was counted as CFU-C. CFU-C counts and cell countsobtained by hemocytometer were multiplied to obtain CFU-C count/1000cells inoculated in the liquid culture.

In Table 1, result of HDEXIg and in Table 2. result of HSEXIg are shown.Experiments were conducted at n=3, values obtained were shown by(mean±SD). In the table, ND means no detection of colony.

TABLE 1 Colony forming cell maintenance action in the long-term liquidculture of human Delta-1 of the present invention Cytokines Week — MIP-1α HDEXIg 0 69 ± 9 68 ± 9 68 ± 9 2 1440 ± 120  720 ± 110 1280 ± 230 4 340± 40 420 ± 80 410 ± 90 6 28 ± 6  96 ± 17 290 ± 60 8 ND ND  88 ± 13

TABLE 2 Colony forming cell maintenance action in the long-term liquidculture of human Serrate-1 of the present invention Cytokines Week —MIP-1 α HSEXIg 0 68 ± 9 68 ± 9 68 ± 9 2 1440 ± 120  720 ± 110 1360 ± 2804 340 ± 40 420 ± 80 560 ± 70 6 28 ± 6  96 ± 17 220 ± 50 8 ND ND 130 ± 50

CFU-C could only be observed until 6^(th) week of cultivation under thecondition without cytokines for maintaining undifferentiated condition,and under the condition with MIP-1α. It could be observed at 8^(th) weekin the presence of HDEXIg or HSEXIg. In comparison with MIP-1α andHDEXIg and HSEXIg. MIP-1α strongly suppressed colony formation at 2weeks of culture, however no suppression in HDEXIg and HSEXIg wereobserved. In maintenance of CFU-C counts at 6 and 8 weeks of culture,HDBXIg and HSEXIg were superior.

Example 12

Effects of HDEXIg and HSEXIg on Liquid Culture of Blood UndifferentiatedCell LTC-IC

In order to observing physiological action of HDEXIg and HSEXIg on theblood undifferentiated cells, umbilical cord blood CD34 positive cellswere cultured for two weeks in the serum-free liquid medium in thepresence of HDEXIg or HSEX1g and known cytokines, and numbers of LTC-IC,which was thought to be most undifferentiated blood cells at presentwere observed.

The umbilical cord blood monocyte CD34 positive cells, 100000 to 20000cells, separated by a method described in Example 10 were cultured inthe following medium for 2 weeks. Numbers of LTC-IC in 4 experimentalgroups, which include a group before cultivation, a group of HDEXIg, agroup of HSEXIg and a control group, were determined. Media used inliquid culture medium were a—medium added with 2% BSA, 10 μg/ml humaninsulin, 200 μg/ml transferrin, 40 μg/ml low density lipoprotein, and10⁻⁵M 2-mercaptoethanol, further added with 100 ng/ml human SCF, 10ng/ml human IL-3, and 100 ng/ml human IL-6. HDEXIg or HSEXIg 1 μg/mlwere added to the above medium. In the control group, human IgG1 wasadded in the equal concentration.

Preparation of human bone marrow stromal cell layer used for LTC-IC, andquantitative assay of frequency of LTC-IC by a limit dilution wereperformed according to a method of Sutherland et al. (Blood, 74, 1563-,1989 and Proc, Natl. Acad. Sci, USA, 87, 3584-, 1990).

The bone marrow mononuclear cells, 1–2×10⁷ cells, obtained in Example 10before the separation and without the silica solution treatment, werecultured in LTC medium (MyeloCul, Stem Cell Technologies Inc., Canada) 5ml added with hydrocortisone 1 μM (Upjohn Japan Co., Japan) in T-25flask (Falcon Inc., U.S.A. ) at 37° C. under 5% carbon dioxide and 100%humidity in the carbon dioxide incubator. Culture was conducted untilthe adhesive cell layers of the stromal cell formation spread more than80% of the bottom area of the culture. Detachment of the cell layer wasperformed by treating with EDTA solution (Cosmobio Co., Japan). Cellswere plated in the 96 well plate (Beckton-Deckinson Inc., U.S.A.), about2×10⁴ cells/well and re-cultivation was continued in the same medium.X-ray, 15Gy, 250 KV peak was irradiated after reconstituted stromal celllayer. Growth of stromal cells was stopped and blood cells in thestromal cells were removed by this treatment. The thus prepared stromalcells were used as stromal cell layer for the experiments.

In the assay of LTC-IC, cell counts in each group were adjusted withinthe ranges of 25–400 cells/well for CD34 positive cells before thecultivation, and 625–20000 cells/well for the cells after thecultivation, and cells were diluted for six step-dilution within theseranges. Each dilution step of cells was co-cultured with the abovestromal cell layer in the 96 well plate, for 16 wells/cells of onedilution step. Culture was performed in the same medium as used instromal formation, at 37° C. under 5% carbon dioxide and 100% humidityin the carbon dioxide gas incubator for 5 weeks. Cells in suspension andin attachment after cultivation were recovered in each well. Collectedcells were transferred to the semi-solid culture medium consisting ofα-medium added with 0.9% methylcellulose, 30% fetal calf serum (FCS, ICNBiomedical Japan), 1% BSA, 10⁻⁵M 2-mercaptoethanol, 100 ng/ml human SCF,10 ng/ml human IL-3, 100 ng/ml human IL-6, 2 U/ml Epo and 10 ng/ml humanG-CSF. After 2 weeks of cultivation, colony forming cells were detectedas the same was as described in Example 10 and 11, and numbers of well,in which colony forming cells were found, were detected. Incidence ofLTC-IC was calculated according to the method of Taswell et al. (J.Immunol. 126, 1614-, 1981) based on the above data.

Graph used for calculation is shown in FIG. 4. In FIG. 4, calculationcurves after liquid culture is shown. A vertical axis shows ratio ofwell for no colonies were observed, and a horizontal axis shows numberof cells/well. In each experimental group, numbers of well, for whichcolonies were not observed, and numbers of cells were plotted, thenregression curve was calculated by the least square method. Number ofcells corresponding to number of 0.37 (a reciprocal of a base of naturallogarithm) for which colonies did not appeared, was calculated. Areciprocal of that number of cells is a frequency of LTC-IC. Further,absolute number of LTC-IC was calculated from initial number of cellsand frequency of LTC-IC.

The result indicated that 243 LTC-IC were found in 25000 cells beforethe liquid culture. In the control group number of cells during 2 weeksof cultivation increased to 1,510,000 cells, and LTC-IC was decreased to49 cells. However, culturing with human Delta-1, i.e. HDEXIg or humanSerrate-1, i.e. HSEXIg, numbers of cells were maintained in 1,310,000and 1,140,000, respectively, and numbers of LTC-IC were slightlydecreased to 115 and 53. Consequently, polypeptide of the presentinvention, especially human Delta-1 could have an activity formaintenance of number of LTC-IC in the liquid culture.

Example 13

Binding of HDEXIg and HSEXIg for Blood Cells

Binding of Notch ligands with various blood cells was studied usingspecific binding of Notch ligands to Notch receptors.

Blood cell lines tested were Jurkat (ATCC TIB-152), Namalwa (ATCCCRL-1432), HL-60 (ATCC CRL-1964), K562 (ATCC CCL-243), THO-1 (ATCC TIB-202), UT-7 (Komatsu et al., Cancer Res., 51, 341–348. 1991), Mo7e (Avanziet al. Br. J. Haematol., 69, 359-, 1988) and CMK (Sato et al. Exp.Hematol., 15, 495–502, 1987). Culturing media for these cells were foundin the reference or ATCC CELL LIMES & HYBRIDOMAS, 8^(th) Ed. (1994).

Cells, 1×10⁶ cells, were suspended in Hank's balanced salt solutioncontaining 2% FCS and 10 mM Hepes. HDEXIg or HSEXIg 1 μg/ml were addedtherein and allowed to stand at 4° C. for overnight. Cells were washedtwice with the Hank's solution. PE labeled sheep anti-human IgGmonoclonal antibody 1 μg/ml was added, allow to stand in ice-cooling for30 minutes, washed twice with the Hank's solution, and suspended in theHank's solution 1 ml. Analysis was performed using the flow cytometer(FACSCalibur). Control groups were used with human IgG1 staining inplace of HDEXIg or HSEXIg staining.

Results are shown in FIG. 5. A vertical axis indicates cell counts and ahorizontal axis indicates fluorescence intensity. Staining with HDEXIgor HSEXIg is shown by solid line and control, a staining with human IgG1is shown by a broken line. In FIG. 5, the left column shows HDEXIg andthe right column shows HSEXIg. As shown in FIG. 5, results indicate thatJurkat: reacted, Namalwa: non-reacted, HL-60: non-reacted, K562:non-reacted, THP-1: non-reacted, UT-7: reacted, Mo7e: non-reacted andCMK: reacted. Since the same results in HDEXIg and HSEXIg were obtained,both recognized the identical molecule and these cells can bedifferentiated.

Effect of the Invention

Notch ligand molecules of the present invention can be used formaintenance of undifferentiated-suppressive substances, and in thepreparation of pharmaceuticals.

1. An isolated antibody that specifically binds with a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 4. 2. The isolatedantibody according to claim 1, wherein the antibody is a polyclonalantibody.
 3. The isolated antibody according to claim 1, wherein theantibody is a monoclonal antibody.
 4. A method for producing anantibody, comprising: a) administering a polypeptide antigen to a hostanimal to induce antibody production against said polypeptide antigen insaid host animal, said polypeptide consisting of the amino acid sequenceof SEQ ID NO: 4; b) monitoring antibody titer produced by saidadministration of said peptide antigen in said host animal; c)extracting antisera produced in said host animal; and d) isolating andselecting at least one antibody from said antisera.
 5. The methodaccording to claim 4, wherein said host animal is a rabbit.
 6. Themethod according to claim 4, wherein said antisera are extracted byaffinity chromatography.